Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Proteomics for understanding miRNA biology

PubMed Central

Huang, Tai-Chung; Pinto, Sneha M.; Pandey, Akhilesh

2013-01-01

MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional regulation of gene expression. Mature miRNAs associate with the RNA interference silencing complex to repress mRNA translation and/or degrade mRNA transcripts. Mass spectrometry-based proteomics has enabled identification of several core components of the canonical miRNA processing pathway and their posttranslational modifications which are pivotal in miRNA regulatory mechanisms. The use of quantitative proteomic strategies has also emerged as a key technique for experimental identification of miRNA targets by allowing direct determination of proteins whose levels are altered because of translational suppression. This review focuses on the role of proteomics and labeling strategies to understand miRNA biology. PMID:23125164

Role of caspase-9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts.

PubMed

Van Ba, Hoa; Hwang, Inho

2014-02-01

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

RNA Interference in Infectious Tropical Diseases

PubMed Central

Hong, Young S.

2008-01-01

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi. PMID:18344671

Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus.

PubMed

Goswami, Suneha; Sahana, Nandita; Pandey, Vanita; Doblas, Paula; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways. Copyright © 2011 Elsevier B.V. All rights reserved.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

PubMed Central

Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

2007-01-01

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

An RNA isolation system for plant tissues rich in secondary metabolites

PubMed Central

2011-01-01

Background Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression. Findings An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel. Conclusions The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization. PMID:21443767

Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum (Coleoptera: Tenebrionidae) revealed by RNA interference.

PubMed

Chen, Q W; Jin, S; Zhang, L; Shen, Q D; Wei, P; Wei, Z M; Wang, S G; Tang, B

2018-06-01

RNA interference (RNAi) is a very effective technique for studying gene function and may be an efficient method for controlling pests. Trehalose-6-phosphate synthase (TPS), which plays a key role in the synthesis of trehalose and insect development, was cloned in Tribolium castaneum (Herbst) (TcTPS) and the putative functions were studied using RNAi via the injection of double-stranded RNA (dsRNA) corresponding to conserved TPS and trehalose-6-phosphate phosphatase domains. Expression analyses show that TcTPS is expressed higher in the fat body, while quantitative real-time polymerase chain reaction results show that the expression of four trehalase isoforms was significantly suppressed by dsTPS injection. Additionally, the expression of six chitin synthesis-related genes, such as hexokinase 2 and glutamine-fructose-6-phosphate aminotransferase, was suppressed at 48 and 72 h post-dsTPS-1 and dsTPS-2 RNA injection, which were two dsTPS fragments that had been designed for two different locations in TcTPS open reading frame, and that trehalose content and trehalase 1 activity decreased significantly at 72 h post-dsRNA injection. Furthermore, T. castaneum injected with dsTPS-1 and dsTPS-2 RNA displayed significantly lower levels of chitin and could not complete the molting process from larvae to pupae, revealing abnormal molting phenotypes. These results demonstrate that silencing TPS gene leads to molting deformities and high mortality rates via regulation of gene expression in the chitin biosynthetic pathway, and may be a promising approach for pest control in the future.

Evaluation and control of miRNA-like off-target repression for RNA interference.

PubMed

Seok, Heeyoung; Lee, Haejeong; Jang, Eun-Sook; Chi, Sung Wook

2018-03-01

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago-miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

Reduction of bilirubin by targeting human heme oxygenase-1 through siRNA.

PubMed

Xia, Zhen-Wei; Li, Chun-E; Jin, You-Xin; Shi, Yi; Xu, Li-Qing; Zhong, Wen-Wei; Li, Yun-Zhu; Yu, Shan-Chang; Zhang, Zi-Li

2007-04-01

Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.

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

NASA Astrophysics Data System (ADS)

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

2004-11-01

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

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

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

PubMed Central

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

2018-01-01

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

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

PubMed

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

2018-03-14

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

[Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133(+) liver cancer stem cells and increases their cisplatin chemosensitivity].

PubMed

Lan, Xi; Wang, Yong; Cao, Shu; Zou, Dongling; Li, Fang; Li, Shaolin

2012-12-01

To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line. CD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry. The sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

Proteomics for understanding miRNA biology.

PubMed

Huang, Tai-Chung; Pinto, Sneha M; Pandey, Akhilesh

2013-02-01

MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional regulation of gene expression. Mature miRNAs associate with the RNA interference silencing complex to repress mRNA translation and/or degrade mRNA transcripts. Mass spectrometry-based proteomics has enabled identification of several core components of the canonical miRNA processing pathway and their posttranslational modifications which are pivotal in miRNA regulatory mechanisms. The use of quantitative proteomic strategies has also emerged as a key technique for experimental identification of miRNA targets by allowing direct determination of proteins whose levels are altered because of translational suppression. This review focuses on the role of proteomics and labeling strategies to understand miRNA biology. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells

PubMed Central

Palanca-Wessels, Maria C.; Booth, Garrett C.; Convertine, Anthony J.; Lundy, Brittany B.; Berguig, Geoffrey Y.; Press, Michael F.; Stayton, Patrick S.; Press, Oliver W.

2016-01-01

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5′ RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5′ RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy. PMID:26840082

Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells.

PubMed

Palanca-Wessels, Maria C; Booth, Garrett C; Convertine, Anthony J; Lundy, Brittany B; Berguig, Geoffrey Y; Press, Michael F; Stayton, Patrick S; Press, Oliver W

2016-02-23

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5' RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5' RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy.

Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation

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

Osabe, Makoto; Sugatani, Junko; Global COE Program, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2more » cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.« less

RNA interference-based therapeutics for inherited long QT syndrome.

PubMed

Li, Guoliang; Ma, Shuting; Sun, Chaofeng

2015-08-01

Inherited long QT syndrome (LQTS) is an electrical heart disorder that manifests with syncope, seizures, and increased risk of torsades de pointes and sudden cardiac death. Dominant-negative current suppression is a mechanism by which pathogenic proteins disrupt the function of ion channels in inherited LQTS. However, current approaches for the management of inherited LQTS are inadequate. RNA interference (RNAi) is a powerful technique that is able to suppress or silence the expression of mutant genes. RNAi may be harnessed to knock out mRNAs that code for toxic proteins, and has been increasingly recognized as a potential therapeutic intervention for a range of conditions. The present study reviews the literature for RNAi-based therapeutics in the treatment of inherited LQTS. Furthermore, this review discusses the combined use of RNAi with the emerging technology of induced pluripotent stem cells for the treatment of inherited LQTS. In addition, key challenges that must be overcome prior to RNAi-based therapies becoming clinically applicable are addressed. In summary, RNAi-based therapy is potentially a powerful therapeutic intervention, although a number of difficulties remain unresolved.

RNA interference-based therapeutics for inherited long QT syndrome

PubMed Central

LI, GUOLIANG; MA, SHUTING; SUN, CHAOFENG

2015-01-01

Inherited long QT syndrome (LQTS) is an electrical heart disorder that manifests with syncope, seizures, and increased risk of torsades de pointes and sudden cardiac death. Dominant-negative current suppression is a mechanism by which pathogenic proteins disrupt the function of ion channels in inherited LQTS. However, current approaches for the management of inherited LQTS are inadequate. RNA interference (RNAi) is a powerful technique that is able to suppress or silence the expression of mutant genes. RNAi may be harnessed to knock out mRNAs that code for toxic proteins, and has been increasingly recognized as a potential therapeutic intervention for a range of conditions. The present study reviews the literature for RNAi-based therapeutics in the treatment of inherited LQTS. Furthermore, this review discusses the combined use of RNAi with the emerging technology of induced pluripotent stem cells for the treatment of inherited LQTS. In addition, key challenges that must be overcome prior to RNAi-based therapies becoming clinically applicable are addressed. In summary, RNAi-based therapy is potentially a powerful therapeutic intervention, although a number of difficulties remain unresolved. PMID:26622327

Efficacy of a Novel Class of RNA Interference Therapeutic Agents

PubMed Central

Matsumoto, Takahiro; D'Alessandro-Gabazza, Corina N.; Gil-Bernabe, Paloma; Boveda-Ruiz, Daniel; Naito, Masahiro; Kobayashi, Tetsu; Toda, Masaaki; Mizutani, Takayuki; Taguchi, Osamu; Morser, John; Eguchi, Yutaka; Kuroda, Masahiko; Ochiya, Takahiro; Hayashi, Hirotake; Gabazza, Esteban C.; Ohgi, Tadaaki

2012-01-01

RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application. PMID:22916145

A Re-Examination of Global Suppression of RNA Interference by HIV-1

PubMed Central

Sanghvi, Viraj R.; Steel, Laura F.

2011-01-01

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells.

PubMed

Tsutsumida, Hideaki; Swanson, Benjamin J; Singh, Pankaj K; Caffrey, Thomas C; Kitajima, Shinichi; Goto, Masamichi; Yonezawa, Suguru; Hollingsworth, Michael A

2006-05-15

MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

PubMed

Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

2017-04-01

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production

PubMed Central

Poenisch, Marion; Metz, Philippe; Blankenburg, Hagen; Ruggieri, Alessia; Lee, Ji-Young; Rupp, Daniel; Rebhan, Ilka; Diederich, Kathrin; Kaderali, Lars; Domingues, Francisco S.; Albrecht, Mario; Lohmann, Volker; Erfle, Holger; Bartenschlager, Ralf

2015-01-01

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses. PMID:25569684

Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals.

PubMed

Qiu, Yang; Xu, Yanpeng; Zhang, Yao; Zhou, Hui; Deng, Yong-Qiang; Li, Xiao-Feng; Miao, Meng; Zhang, Qiang; Zhong, Bo; Hu, Yuanyang; Zhang, Fu-Chun; Wu, Ligang; Qin, Cheng-Feng; Zhou, Xi

2017-06-20

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

A simple and robust vector-based shRNA expression system used for RNA interference.

PubMed

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

Abacavir coadministration does not interfere with the suppressive activity of ribavirin in an HCV replicon system.

PubMed

Van den Eynde, Eva; Quer, Josep; Cubero, María; Curran, Adriá; Homs, María; Garcia-Cehic, Damir; Falco, Vicenç; Ribera, Esteban; Esteban, Juan I; Pahissa, Albert; Crespo, Manuel

2011-01-01

HCV is a major cause of morbidity and mortality in HIV-coinfected patients. Several observational studies have suggested that HCV response to pegylated interferon and ribavirin is lower in HIV-coinfected patients treated with abacavir. It has been postulated that abacavir could compete with ribavirin to be phosphorylated, leading to a reduction in the active form of the drug (triphosphorylated ribavirin). Here, we studied the effect of abacavir, tenofovir or lamivudine addition on the suppressive activity of ribavirin in an HCV RNA replicon system. We used the human hepatoma HuH-7 cell clone 9B containing the HCV genotype 1b replicon I389/NS3-3'. Cells were treated for 24 h with ribavirin (0, 10 and 50 μM) plus abacavir, tenofovir or lamivudine at doses of 0, 10 and 50 μM and HCV RNA production was quantified by real-time PCR in triplicate assays. Results were expressed as mean±SD of the HCV RNA produced per cell (log(10) IU/cell). Means were compared using the Student's t-test. Ribavirin treatment produced a dose-dependent suppression of HCV RNA production by the replicon system. Combination of ribavirin and interferon resulted in an additive antiviral activity. The addition of abacavir did not modify the suppressive activity of ribavirin on the replicon HCV RNA expression. Similar results were obtained when ribavirin was used in combination with tenofovir or lamivudine. In a subgenomic HCV RNA replicon system, the antiviral effect of ribavirin was not modified by the addition of abacavir, tenofovir or lamivudine. © 2011 International Medical Press

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

PubMed

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

2013-08-01

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

Postexposure Protection of Guinea Pigs against a Lethal Ebola Virus Challenge is Conferred by RNA Interference

DTIC Science & Technology

2006-06-15

because its suppression should lead to a nearly total loss of all RNA synthesis , but also because of the absence of similar proteins in mammalian cells...protects mice from fulminant hepatitis. Nat Med 2003; 9:347–51. 12. Ge Q, Filip L, Bai A, Nguyen T, Eisen HN, Chen J. Inhibition of influenza virus...human beta interferon gene in simian cells defective in interferon synthesis . Mol Cell Biol 1986; 6:2279–83. 21. Spann KM, Tran KC, Collins PL

RNA Interference Using c-Myc-Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models.

PubMed

Tangudu, Naveen K; Verma, Vinod K; Clemons, Tristan D; Beevi, Syed S; Hay, Trevor; Mahidhara, Ganesh; Raja, Meera; Nair, Rekha A; Alexander, Liza E; Patel, Anant B; Jose, Jedy; Smith, Nicole M; Zdyrko, Bogdan; Bourdoncle, Anne; Luzinov, Igor; Iyer, K Swaminathan; Clarke, Alan R; Dinesh Kumar, Lekha

2015-05-01

In this article, we report the development and preclinical validation of combinatorial therapy for treatment of cancers using RNA interference (RNAi). RNAi technology is an attractive approach to silence genes responsible for disease onset and progression. Currently, the critical challenge facing the clinical success of RNAi technology is in the difficulty of delivery of RNAi inducers, due to low transfection efficiency, difficulties of integration into host DNA and unstable expression. Using the macromolecule polyglycidal methacrylate (PGMA) as a platform to graft multiple polyethyleneimine (PEI) chains, we demonstrate effective delivery of small oligos (anti-miRs and mimics) and larger DNAs (encoding shRNAs) in a wide variety of cancer cell lines by successful silencing/activation of their respective target genes. Furthermore, the effectiveness of this therapy was validated for in vivo tumor suppression using two transgenic mouse models; first, tumor growth arrest and increased animal survival was seen in mice bearing Brca2/p53-mutant mammary tumors following daily intratumoral treatment with nanoparticles conjugated to c-Myc shRNA. Second, oral delivery of the conjugate to an Apc-deficient crypt progenitor colon cancer model increased animal survival and returned intestinal tissue to a non-wnt-deregulated state. This study demonstrates, through careful design of nonviral nanoparticles and appropriate selection of therapeutic gene targets, that RNAi technology can be made an affordable and amenable therapy for cancer. ©2015 American Association for Cancer Research.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.

PubMed

Parisien, Jean-Patrick; Lenoir, Jessica J; Mandhana, Roli; Rodriguez, Kenny R; Qian, Kenin; Bruns, Annie M; Horvath, Curt M

2018-06-01

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG-I and MDA5 stimulate a mitochondria-localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA LGP2 associates with the C-terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C-terminal domain, and LGP2 can regulate TRAF-mediated signaling pathways in trans , including IL-1β, TNFα, and cGAMP These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response. © 2018 The Authors.

Knockdown of p53 suppresses Nanog expression in embryonic stem cells

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

Abdelalim, Essam Mohamed, E-mail: emohamed@qf.org.qa; Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192; Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia

2014-01-10

Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21more » and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.« less

Convergent transcription in the butyrolactone regulon in Streptomyces coelicolor confers a bistable genetic switch for antibiotic biosynthesis.

PubMed

Chatterjee, Anushree; Drews, Laurie; Mehra, Sarika; Takano, Eriko; Kaznessis, Yiannis N; Hu, Wei-Shou

2011-01-01

cis-encoded antisense RNAs (cis asRNA) have been reported to participate in gene expression regulation in both eukaryotic and prokaryotic organisms. Its presence in Streptomyces coelicolor has also been reported recently; however, its role has yet to be fully investigated. Using mathematical modeling we explore the role of cis asRNA produced as a result of convergent transcription in scbA-scbR genetic switch. scbA and scbR gene pair, encoding repressor-amplifier proteins respectively, mediates the synthesis of a signaling molecule, the γ-butyrolactone SCB1 and controls the onset of antibiotic production. Our model considers that transcriptional interference caused by convergent transcription of two opposing RNA polymerases results in fatal collision and transcriptional termination, which suppresses transcription efficiency. Additionally, convergent transcription causes sense and antisense interactions between complementary sequences from opposing strands, rendering the full length transcript inaccessible for translation. We evaluated the role of transcriptional interference and the antisense effect conferred by convergent transcription on the behavior of scbA-scbR system. Stability analysis showed that while transcriptional interference affects the system, it is asRNA that confers scbA-scbR system the characteristics of a bistable switch in response to the signaling molecule SCB1. With its critical role of regulating the onset of antibiotic synthesis the bistable behavior offers this two gene system the needed robustness to be a genetic switch. The convergent two gene system with potential of transcriptional interference is a frequent feature in various genomes. The possibility of asRNA regulation in other such gene-pairs is yet to be examined.

A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

PubMed Central

Feng, Hui; Beck, Jürgen; Nassal, Michael; Hu, Kang-hong

2011-01-01

Background The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. Methodology/Principal Findings Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. Conclusions/Significance This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B. PMID:22125633

Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer.

PubMed

Lan, Yang; Xiao, Xuewei; He, Zhengchi; Luo, Yu; Wu, Chuanfang; Li, Ling; Song, Xu

2018-06-20

Overexpressed in colon carcinoma-1 (OCC-1) is one of the earliest annotated long noncoding RNAs (lncRNAs) in colorectal cancer (CRC); however, its function remains largely unknown. Here, we revealed that OCC-1 plays a tumor suppressive role in CRC. OCC-1 knockdown by RNA interference promotes cell growth both in vitro and in vivo, which is largely due to its ability to inhibit G0 to G1 and G1 to S phase cell cycle transitions. In addition, overexpression of OCC-1 can suppress cell growth in OCC-1 knockdown cells. OCC-1 exerts its function by binding to and destabilizing HuR (ELAVL1), a cancer-associated RNA binding protein (RBP) which can bind to and stabilize thousands of mRNAs. OCC-1 enhances the binding of ubiquitin E3 ligase β-TrCP1 to HuR and renders HuR susceptible to ubiquitination and degradation, thereby reducing the levels of HuR and its target mRNAs, including the mRNAs directly associated with cancer cell growth. These findings reveal that lncRNA OCC-1 can regulate the levels of a large number of mRNAs at post-transcriptional level through modulating RBP HuR stability.

Suppression of Bedbug’s Reproduction by RNA Interference of Vitellogenin

PubMed Central

Moriyama, Minoru; Hosokawa, Takahiro; Tanahashi, Masahiko; Nikoh, Naruo; Fukatsu, Takema

2016-01-01

Recent resurgence of the bedbug Cimex lectularius is a global problem on the public health. On account of the worldwide rise of insecticide-resistant bedbug populations, exploration of new approaches to the bedbug control and management is anticipated. In this context, gene silencing by RNA interference (RNAi) has been considered for its potential application to pest control and management, because RNAi enables specific suppression of target genes and thus flexible selection of target traits to be disrupted. In this study, in an attempt to develop a control strategy targeting reproduction of the bedbug, we investigated RNAi-mediated gene silencing of vitellogenin (Vg), a major yolk protein precursor essential for oogenesis. From the bedbug transcriptomes, we identified a typical Vg gene and a truncated Vg gene, which were designated as ClVg and ClVg-like, respectively. ClVg gene was highly expressed mainly in the fat body of adult females, which was more than 100 times higher than the expression level of ClVg-like gene, indicating that ClVg gene is the primary functional Vg gene in the bedbug. RNAi-mediated suppression of ClVg gene expression in adult females resulted in drastically reduced egg production, atrophied ovaries, and inflated abdomen due to hypertrophied fat bodies. These phenotypic consequences are expected not only to suppress the bedbug reproduction directly but also to deteriorate its feeding and survival indirectly via behavioral modifications. These results suggest the potential of ClVg gene as a promising target for RNAi-based population management of the bedbug. PMID:27096422

Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

PubMed Central

Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

2011-01-01

Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

Widespread suppression of huntingtin with convection-enhanced delivery of siRNA.

PubMed

Stiles, David K; Zhang, Zhiming; Ge, Pei; Nelson, Brian; Grondin, Richard; Ai, Yi; Hardy, Peter; Nelson, Peter T; Guzaev, Andrei P; Butt, Mark T; Charisse, Klaus; Kosovrasti, Verbena; Tchangov, Lubomir; Meys, Michael; Maier, Martin; Nechev, Lubomir; Manoharan, Muthiah; Kaemmerer, William F; Gwost, Douglas; Stewart, Gregory R; Gash, Don M; Sah, Dinah W Y

2012-01-01

Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease. Copyright © 2011 Elsevier Inc. All rights reserved.

A Small GTP-Binding Host Protein Is Required for Entry of Powdery Mildew Fungus into Epidermal Cells of Barley1

PubMed Central

Schultheiss, Holger; Dechert, Cornelia; Kogel, Karl-Heinz; Hückelhoven, Ralph

2002-01-01

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions. PMID:11950993

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

PubMed Central

Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

2013-01-01

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

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.

The PPARdelta agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibition.

PubMed

Kino, T; Rice, K C; Chrousos, G P

2007-05-01

Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor delta (PPARdelta), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. We examined the ability of the synthetic PPARdelta agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein alpha1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of alpha2-acid glycoprotein, beta-fibrinogen and alpha2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPARdelta receptor, but not of PPARalpha or gamma, attenuated the suppressive effect of GW501516 on interleukin-6-induced alpha1-antichymotrypsin mRNA expression, indicating that PPARdelta specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the alpha1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPARdelta on this promoter. Overexpression of PPARdelta enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the alpha1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. These findings indicate that agonist-activated PPARdelta interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPARdelta agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role.

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

PubMed

Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

2015-12-01

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

PubMed

Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

2018-02-12

Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

RNA major groove modifications improve siRNA stability and biological activity

PubMed Central

Terrazas, Montserrat; Kool, Eric T.

2009-01-01

RNA 5-methyl and 5-propynyl pyrimidine analogs were substituted into short interfering RNAs (siRNAs) to probe major groove steric effects in the active RNA-induced silencing complex (RISC). Synthetic RNA guide strands containing varied combinations of propynyl and methyl substitution revealed that all C-5 substitutions increased the thermal stability of siRNA duplexes containing them. Cellular gene suppression experiments using luciferase targets in HeLa cells showed that the bulky 5-propynyl modification was detrimental to RNA interference activity, despite its stabilization of the helix. Detrimental effects of this substitution were greatest at the 5′-half of the guide strand, suggesting close steric approach of proteins in the RISC complex with that end of the siRNA/mRNA duplex. However, substitutions with the smaller 5-methyl group resulted in gene silencing activities comparable to or better than that of wild-type siRNA. The major groove modifications also increased the serum stability of siRNAs. PMID:19042976

Suppression of allene oxide synthase 3 in potato increases degree of arbuscular mycorrhizal fungal colonization.

PubMed

Morcillo, Rafael Jorge León; Navarrete, María Isabel Tamayo; Bote, Juan Antonio Ocampo; Monguio, Salomé Prat; García-Garrido, José Manuel

2016-01-15

Arbuscular mycorrhizal (AM) is a mutually beneficial interaction among higher plants and soil fungi of the phylum Glomeromycota. Numerous studies have pointed that jasmonic acid plays an important role in the development of the intraradical fungus. This compound belongs to a group of biologically active compounds known as oxylipins which are derived from the oxidative metabolism of polyunsaturated fatty acids. Studies of the regulatory role played by oxylipins in AM colonization have generally focused on jasmonates, while few studies exist on the 9-LOX pathway of oxylipins during AM formation. Here, the cDNA of Allene oxide synthase 3 (AOS3), a key enzyme in the 9-LOX pathway, was used in the RNA interference (RNAi) system to transform potato plants in order to suppress its expression. Results show increases in AOS3 gene expression and 9-LOX products in roots of wild type potato mycorrhizal plants. The suppression of AOS3 gene expression increases the percentage of root with mycorrhizal colonization at early stages of AM formation. AOS3 RNA interference lead to an induction of LOXA and 13-LOX genes, a reduction in AOS3 derived 9-LOX oxylipin compounds and an increase in jasmonic acid content, suggesting compensation between 9 and 13-LOX pathways. The results in a whole support the hypothesis of a regulatory role for the 9-LOX oxylipin pathway during mycorrhization. Copyright © 2015 Elsevier GmbH. All rights reserved.

Biomimetic RNA-silencing nanocomplexes: overcoming multidrug resistance in cancer cells.

PubMed

Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang; Yang, Min; Chen, Xiaoyuan

2014-02-10

RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by an RNA-induced silencing complex (RISC). Herein, we present a synthetic RISC-mimic nanocomplex, which can actively cleave its target RNA in a sequence-specific manner. With high enzymatic stability and efficient self-delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. These nanocomplexes, which target multidrug resistance, are not only able to bypass the P-glycoprotein (Pgp) transporter, due to their nano-size effect, but also effectively suppress Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp-transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Banerjee, Sagar; Banerjee, Anamika; Gill, Sarvajeet S; Gupta, Om P; Dahuja, Anil; Jain, Pradeep K; Sirohi, Anil

2017-01-01

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

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

PubMed Central

Hsu, Shih-Feng; Su, Wen-Chi; Jeng, King-Song

2015-01-01

ABSTRACT Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection. PMID:25589657

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

PubMed

Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

2012-08-01

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

Study on Interference Suppression Algorithms for Electronic Noses: A Review

PubMed Central

Liang, Zhifang; Zhang, Ci; Sun, Hao; Liu, Tao

2018-01-01

Electronic noses (e-nose) are composed of an appropriate pattern recognition system and a gas sensor array with a certain degree of specificity and broad spectrum characteristics. The gas sensors have their own shortcomings of being highly sensitive to interferences which has an impact on the detection of target gases. When there are interferences, the performance of the e-nose will deteriorate. Therefore, it is urgent to study interference suppression techniques for e-noses. This paper summarizes the sources of interferences and reviews the advances made in recent years in interference suppression for e-noses. According to the factors which cause interference, interferences can be classified into two types: interference caused by changes of operating conditions and interference caused by hardware failures. The existing suppression methods were summarized and analyzed from these two aspects. Since the interferences of e-noses are uncertain and unstable, it can be found that some nonlinear methods have good effects for interference suppression, such as methods based on transfer learning, adaptive methods, etc. PMID:29649152

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation

PubMed Central

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

2004-01-01

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation. PMID:15084750

Down-regulated long non-coding RNA-ATB in preeclampsia and its effect on suppressing migration, proliferation, and tube formation of trophoblast cells.

PubMed

Liu, Xijing; Chen, Hongqin; Kong, Weiqi; Zhang, Yanping; Cao, Liyuan; Gao, Linbo; Zhou, Rong

2017-01-01

Preeclampsia is a pregnancy-specific syndrome and is one of the main causes of maternal, fetal, and neonatal morbidity and mortality. Inadequate trophoblast invasion and failure of uterine spiral artery remodeling exert a major role in the development of preeclampsia, especially the early-onset one. LncRNA-ATB is verified to be aberrantly expressed in many cancers and promote the invasion-metastasis and proliferation cascades. But little is known of lncRNA-ATB's role in preeclampsia. The aim of current study is to identify the changes of lncRNA-ATB in preeclampsia and its effects on trophoblast. The lncRNA-ATB levels were decreased in placental samples collected from preeclampsia women (n = 51) compared to those of healthy pregnant women (n = 40) by qRT-PCR analysis. Besides, it is demonstrated that lncRNA-ATB was intense stained in the trophoblast of the placenta by performing in-situ hybridization. By designing RNA interference species to suppress lncRNA-ATB and specific plasmids designed to overexpress lncRNA-ATB, we identify the role of lncRNA-ATB on the functions of trophoblast cell-line, HTR-8/SVneo. Inhibition of endogenous lncRNA-ATB decreased migration, proliferation, tube-formation of HTR-8/SVneo cells. In addition, overexpression of lncRNA-ATB promoted migration, proliferation, and tube-formation of HTR-8/SVneo cells. Therefore, lncRNA-ATB might be involved in the pathogenesis of preeclampsia by regulating the process of trophoblast invasion and endovascular formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Down-regulation of a chitin synthase a gene by RNA interference enhances pathogenicity of Beauveria bassiana ANU1 against Spodoptera exigua (HÜBNER).

PubMed

Lee, Jung-Bok; Kim, Hyun Soo; Park, Youngjin

2017-02-01

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS-A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS-B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS-A gene from the beet armyworm, Spodoptera exigua (SeCHS-A). The SeCHS-A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS-A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real-time-PCR analysis. Expression of SeCHS-A gene was suppressed by feeding double-stranded RNA (dsCHS-A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS-A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS-A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS-A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana. © 2017 Wiley Periodicals, Inc.

RNA interference (RNAI) as a tool to engineer high nutritional value in chicory (Chicorium intybus).

PubMed

Asad, M

2006-01-01

The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.

Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy.

PubMed

Yang, Xian-Zhu; Dou, Shuang; Sun, Tian-Meng; Mao, Cheng-Qiong; Wang, Hong-Xia; Wang, Jun

2011-12-10

Delivery of small interfering RNA (siRNA) has been one of the major hurdles for the application of RNA interference in therapeutics. Here, we describe a cationic lipid assisted polymeric nanoparticle system with stealthy property for efficient siRNA encapsulation and delivery, which was fabricated with poly(ethylene glycol)-b-poly(d,l-lactide), siRNA and a cationic lipid, using a double emulsion-solvent evaporation technique. By incorporation of the cationic lipid, the encapsulation efficiency of siRNA into the nanoparticles could be above 90% and the siRNA loading weight ratio was up to 4.47%, while the diameter of the nanoparticles was around 170 to 200nm. The siRNA retained its integrity within the nanoparticles, which were effectively internalized by cancer cells and escaped from the endosome, resulting in significant gene knockdown. This effect was demonstrated by significant down-regulation of luciferase expression in HepG2-luciferase cells which stably express luciferase, and suppression of polo-like kinase 1 (Plk1) expression in HepG2 cells, following delivery of specific siRNAs by the nanoparticles. Furthermore, the nanoparticles carrying siRNA targeting the Plk1 gene were found to induce remarkable apoptosis in both HepG2 and MDA-MB-435s cancer cells. Systemic delivery of specific siRNA by nanoparticles significantly inhibited luciferase expression in an orthotopic murine liver cancer model and suppressed tumor growth in a MDA-MB-435s murine xenograft model, suggesting its therapeutic promise in disease treatment. Copyright © 2011 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-09-15

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

Prediction of effective RNA interference targets and pathway-related genes in lepidopteran insects by RNA sequencing analysis.

PubMed

Guan, Ruo-Bing; Li, Hai-Chao; Miao, Xue-Xia

2018-06-01

When using RNA interference (RNAi) to study gene functions in Lepidoptera insects, we discovered that some genes could not be suppressed; instead, their expression levels could be up-regulated by double-stranded RNA (dsRNA). To predict which genes could be easily silenced, we treated the Asian corn borer (Ostrinia furnacalis) with dsGFP (green fluorescent protein) and dsMLP (muscle lim protein). A transcriptome sequence analysis was conducted using the cDNAs 6 h after treatment with dsRNA. The results indicated that 160 genes were up-regulated and 44 genes were down-regulated by the two dsRNAs. Then, 50 co-up-regulated, 25 co-down-regulated and 43 unaffected genes were selected to determine their RNAi responses. All the 25 down-regulated genes were knocked down by their corresponding dsRNA. However, several of the up-regulated and unaffected genes were up-regulated when treated with their corresponding dsRNAs instead of being knocked down. The genes up-regulated by the dsGFP treatment may be involved in insect immune responses or the RNAi pathway. When the immune-related genes were excluded, only seven genes were induced by dsGFP, including ago-2 and dicer-2. These results not only provide a reference for efficient RNAi target predications, but also provide some potential RNAi pathway-related genes for further study. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Reversal of multidrug resistance in MCF-7/Adr cells by codelivery of doxorubicin and BCL2 siRNA using a folic acid-conjugated polyethylenimine hydroxypropyl-β-cyclodextrin nanocarrier

PubMed Central

Li, Jin-Ming; Zhang, Wei; Su, Hua; Wang, Yuan-Yuan; Tan, Cai-Ping; Ji, Liang-Nian; Mao, Zong-Wan

2015-01-01

Systemic administration of chemotherapy for cancer often faces drug resistance, limiting its applications in cancer therapy. In this study, we developed a simple multifunctional nanocarrier based on polyethylenimine (PEI) to codeliver doxorubicin (DOX) and BCL2 small interfering RNA (siRNA) for overcoming multidrug resistance (MDR) and enhancing apoptosis in MCF-7/Adr cancer cells by combining chemotherapy and RNA interference (RNAi) therapy. The low-molecular-weight branch PEI was used to conjugate hydroxypropyl-β-cyclodextrin (HP-β-CD) and folic acid (FA), forming the codelivery nanocarrier (FA-HP-β-CD-PEI) to encapsulate DOX with the cavity HP-β-CD and bind siRNA with the positive charge of PEI for tumor-targeting codelivering drugs. The drug-loaded nanocomplexes (FA-HP-β-CD-PEI/DOX/siRNA) showed uniform size distribution, high cellular uptake, and significant gene suppression of BCL2, displaying the potential of overcoming MDR for enhancing the effect of anticancer drugs. Furthermore, the nanocomplexes achieved significant cell apoptosis through a mechanism of downregulating the antiapoptotic protein BCL2, resulted in improving therapeutic efficacy of the coadministered DOX by tumor targeting and RNA interference. Our study indicated that combined RNAi therapy and chemotherapy using our functional codelivery nanocarrier could overcome MDR and enhance apoptosis in MDR cancer cells for a potential application in treating MDR cancers. PMID:25960653

Gene therapy in animal models of autosomal dominant retinitis pigmentosa

PubMed Central

Rossmiller, Brian; Mao, Haoyu

2012-01-01

Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success. PMID:23077406

Glutaredoxin modulates platelet-derived growth factor-dependent cell signaling by regulating the redox status of low molecular weight protein-tyrosine phosphatase.

PubMed

Kanda, Munetake; Ihara, Yoshito; Murata, Hiroaki; Urata, Yoshishige; Kono, Takaaki; Yodoi, Junji; Seto, Shinji; Yano, Katsusuke; Kondo, Takahito

2006-09-29

Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) beta was suppressed in GRX gene-transfected cells, compared with controls. Conversely, the phosphorylation was enhanced by depletion of GRX by RNA interference. In this study we focused on the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in the dephosphorylation of PDGFRbeta via a redox-dependent mechanism. We found that depletion of LMW-PTP using RNA interference enhanced the PDGF-BB-induced phosphorylation of PDGFRbeta, indicating that LMW-PTP works for PDGFRbeta. The enhancement of the phosphorylation of PDGFRbeta was well correlated with inactivation of LMW-PTP by cellular peroxide generated in the cells stimulated with PDGF-BB. In vitro, with hydrogen peroxide treatment, LMW-PTP showed decreased activity with the concomitant formation of dithiothreitol-reducible oligomers. GRX protected LMW-PTP from hydrogen peroxide-induced oxidation and inactivation in concert with glutathione, NADPH, and glutathione disulfide reductase. This strongly suggests that retention of activity of LMW-PTP by enhanced GRX expression suppresses the proliferation of cells treated with PDGF-BB via enhanced dephosphorylation of PDGFRbeta. Thus, GRX plays an important role in PDGF-BB-dependent cell proliferation by regulating the redox state of LMW-PTP.

Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys?

PubMed

Block, Anna; Alfano, James R

2011-02-01

The phytopathogenic bacterium Pseudomonas syringae can suppress both pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) by the injection of type III effector (T3E) proteins into host cells. T3Es achieve immune suppression using a variety of strategies including interference with immune receptor signaling, blocking RNA pathways and vesicle trafficking, and altering organelle function. T3Es can be recognized indirectly by resistance proteins monitoring specific T3E targets resulting in ETI. It is presently unclear whether the monitored targets represent bona fide virulence targets or guarded decoys. Extensive overlap between PTI and ETI signaling suggests that T3Es may suppress both pathways through common targets and by possessing multiple activities. Copyright © 2010 Elsevier Ltd. All rights reserved.

beta-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures.

PubMed

Ye, Ping; Hu, Qichen; Liu, Hedi; Yan, Yun; D'ercole, A Joseph

2010-07-01

By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether beta-catenin, a molecule that is a downstream target of glycogen synthase kinase-3beta (GSK3beta) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases beta-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3beta. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in beta-catenin and cyclin D1 mRNA, while suppression of GSK3beta activity simulated IGF-I actions. Knocking-down beta-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that beta-catenin is an important downstream molecule in the PI3-Akt-GSK3beta pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. Copyright 2010 Wiley-Liss, Inc.

RNA interference of three up-regulated transcripts associated with insecticide resistance in an imidacloprid resistant population of Leptinotarsa decemlineata.

PubMed

Clements, Justin; Schoville, Sean; Peterson, Nathan; Huseth, Anders S; Lan, Que; Groves, Russell L

2017-01-01

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a major agricultural pest of potatoes in the Central Sands production region of Wisconsin. Previous studies have shown that populations of L. decemlineata have become resistant to many classes of insecticides, including the neonicotinoid insecticide, imidacloprid. Furthermore, L. decemlineata has multiple mechanisms of resistance to deal with a pesticide insult, including enhanced metabolic detoxification by cytochrome p450s and glutathione S-transferases. With recent advances in the transcriptomic analysis of imidacloprid susceptible and resistant L. decemlineata populations, it is possible to investigate the role of candidate genes involved in imidacloprid resistance. A recently annotated transcriptome analysis of L. decemlineata was obtained from select populations of L. decemlineata collected in the Central Sands potato production region, which revealed a subset of mRNA transcripts constitutively up-regulated in resistant populations. We hypothesize that a portion of the up-regulated transcripts encoding for genes within the resistant populations also encode for pesticide resistance and can be suppressed to re-establish a susceptible phenotype. In this study, a discrete set of three up-regulated targets were selected for RNA interference experiments using a resistant L. decemlineata population. Following the successful suppression of transcripts encoding for a cytochrome p450, a cuticular protein, and a glutathione synthetase protein in a select L. decemlineata population, we observed reductions in measured resistance to imidacloprid that strongly suggest these genes control essential steps in imidacloprid metabolism in these field populations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Therapeutic synergy between microRNA and siRNA in ovarian cancer treatment.

PubMed

Nishimura, Masato; Jung, Eun-Jung; Shah, Maitri Y; Lu, Chunhua; Spizzo, Riccardo; Shimizu, Masayoshi; Han, Hee Dong; Ivan, Cristina; Rossi, Simona; Zhang, Xinna; Nicoloso, Milena S; Wu, Sherry Y; Almeida, Maria Ines; Bottsford-Miller, Justin; Pecot, Chad V; Zand, Behrouz; Matsuo, Koji; Shahzad, Mian M; Jennings, Nicholas B; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Sood, Anil K; Calin, George A

2013-11-01

Development of improved RNA interference-based strategies is of utmost clinical importance. Although siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA (miRNA) further "boosts" its antitumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EphA2 siRNA showed synergistic antitumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is at least in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA-siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases. This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA in nanoliposomal particles to target oncogenic pathways altered in ovarian cancer. Combined targeting of the Eph pathway using EphA2-targeting siRNA and the tumor suppressor miR-520d-3p exhibits remarkable therapeutic synergy and enhanced tumor suppression in vitro and in vivo compared with either monotherapy alone. ©2013 AACR.

Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

PubMed Central

Chu, Chia-ying

2006-01-01

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression. PMID:16756390

Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

PubMed

Chen, Y; Redinbaugh, M G; Michel, A P

2015-06-01

Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G. nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G. nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G. nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control. © 2015 The Royal Entomological Society.

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

NASA Astrophysics Data System (ADS)

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

2004-04-01

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

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.

G-protein gamma subunit 1 is required for sugar reception in Drosophila

PubMed Central

Ishimoto, Hiroshi; Takahashi, Kuniaki; Ueda, Ryu; Tanimura, Teiichi

2005-01-01

Though G-proteins have been implicated in the primary step of taste signal transduction, no direct demonstration has been done in insects. We show here that a G-protein gamma subunit, Gγ1, is required for the signal transduction of sugar taste reception in Drosophila. The Gγ1 gene is expressed mainly in one of the gustatory receptor neurons. Behavioral responses of the flies to sucrose were reduced by the targeted suppression of neural functions of Gγ1-expressing cells using neural modulator genes such as the modified Shaker K+ channel (EKO), the tetanus toxin light chain or the shibire (shits1) gene. RNA interference targeting to the Gγ1 gene reduced the amount of Gγ1 mRNA and suppressed electrophysiological response of the sugar receptor neuron. We also demonstrated that responses to sugars were lowered in Gγ1 null mutant, Gγ1N159. These results are consistent with the hypothesis that Gγ1 participates in the signal transduction of sugar taste reception. PMID:16121192

SIRT1 suppresses adipogenesis by activating Wnt/β-catenin signaling in vivo and in vitro

PubMed Central

Zhou, Yuanfei; Song, Tongxing; Peng, Jie; Zhou, Zheng; Wei, Hongkui; Zhou, Rui; Jiang, Siwen; Peng, Jian

2016-01-01

Sirtuin 1 (SIRT1) regulates adipocyte and osteoblast differentiation. However, the underlying mechanism should be investigated. This study revealed that SIRT1 acts as a crucial repressor of adipogenesis. RNA-interference-mediated SIRT1 knockdown or genetic ablation enhances adipogenic potential, whereas SIRT1 overexpression inhibits adipogenesis in mesenchymal stem cells (MSCs). SIRT1 also deacetylates the histones of sFRP1, sFRP2, and Dact1 promoters; inhibits the mRNA expression of sFRP1, sFRP2, and Dact1; activates Wnt signaling pathways; and suppresses adipogenesis. SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus and thus induces the transcription of genes that block MSC adipogenesis. In mice, the partial absence of SIRT1 promotes the formation of white adipose tissues without affecting the development of the body of mice. Our study described the regulatory role of SIRT1 in Wnt signaling and proposed a regulatory mechanism of adipogenesis. PMID:27776347

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

NASA Astrophysics Data System (ADS)

Travella, Silvia; Keller, Beat

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

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

PubMed

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

2010-05-01

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

Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella

PubMed Central

Hameed, Muhammad Salman; Wang, Zhengbing; Yang, Guang

2018-01-01

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella (PxAgo3) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella. PMID:29677157

Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella.

PubMed

Hameed, Muhammad Salman; Wang, Zhengbing; Vasseur, Liette; Yang, Guang

2018-04-20

Argonaute (Ago) protein family plays a key role in the RNA interference (RNAi) process in different insects including Lepidopteran. However, the role of Ago proteins in the RNAi pathway of Plutella xylostella is still unknown. We cloned an Argonaute3 gene in P. xylostella ( PxAgo3 ) with the complete coding sequence of 2832 bp. The encoded protein had 935 amino acids with an expected molecular weight of 108.9 kDa and an isoelectric point of 9.29. It contained a PAZ (PIWI/Argonaute/Zwile) domain and PIWI (P-element-induced whimpy testes) domain. PxAgo3 was classified into the Piwi subfamily of Ago proteins with a high similarity of 93.0% with Bombyx mori Ago3 (BmAgo3). The suppression of PxAgo3 by dsPxAgo3 was observed 3 h after treatment and was maintained until 24 h. Knockdown of PxAgo3 decreased the suppression level of PxActin by dsPxActin in P. xylostella cells, while overexpression of PxAgo3 increased the RNAi efficiency. Our results suggest that PxAgo3 play a key role in the double stranded RNA (dsRNA)-regulated RNAi pathway in P. xylostella .

FGF21 regulates melanogenesis in alpaca melanocytes via ERK1/2-mediated MITF downregulation.

PubMed

Wang, Ruiwei; Chen, Tianzhi; Zhao, Bingling; Fan, Ruiwen; Ji, Kaiyuan; Yu, Xiuju; Wang, Xianjun; Dong, Changsheng

2017-08-19

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production. Copyright © 2017. Published by Elsevier Inc.

Engineering N-Glycosylation Pathway in Insect Cells: Suppression of β-N-Acetylglucosaminidase and Expression of β-1,4-Galactosyltransferase.

PubMed

Kim, Yeon Kyu; Cha, Hyung Joon

2015-01-01

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of β(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

47 CFR 80.217 - Suppression of interference aboard ships.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 5 2012-10-01 2012-10-01 false Suppression of interference aboard ships. 80.217 Section 80.217 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL... interference aboard ships. (a) A voluntarily equipped ship station receiver must not cause harmful interference...

47 CFR 80.217 - Suppression of interference aboard ships.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 5 2014-10-01 2014-10-01 false Suppression of interference aboard ships. 80.217 Section 80.217 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL... interference aboard ships. (a) A voluntarily equipped ship station receiver must not cause harmful interference...

47 CFR 80.217 - Suppression of interference aboard ships.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 5 2013-10-01 2013-10-01 false Suppression of interference aboard ships. 80.217 Section 80.217 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL... interference aboard ships. (a) A voluntarily equipped ship station receiver must not cause harmful interference...

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Cue-independent forgetting by intentional suppression - Evidence for inhibition as the mechanism of intentional forgetting.

PubMed

Wang, Yingying; Cao, Zhijun; Zhu, Zijian; Cai, Huaqian; Wu, Yanhong

2015-10-01

People are able to intentionally forget unwanted memories through voluntary suppression, as revealed by the Think/No-think (TNT) paradigm. However, the nature of intentional forgetting is controversial. Findings that forgetting is independent of retrieval cues suggest that inhibitory control underlies intentional forgetting, but this result is also in line with an interference account. To resolve this controversy, we have directly contrasted the cue-independent characteristic of suppression versus interference. A double-cue paradigm was used, in which two different cues were associated with the same target during initial memory formation. Only one cue-target association received further interference/suppression training. In the test phase, when both cues were used to retrieve the target, we found that interference caused memory impairment that was restricted to the trained cue-target association, while suppression induced forgetting that generalized to the independent cue-target association. Therefore, the effect of suppression differs from that of interference. The cue-independent forgetting by voluntary suppression indicates that the target memory itself is inhibited, providing evidence that the underlying mechanism of suppression-induced forgetting is inhibitory control. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation and function of DNA methylation in plants and animals

PubMed Central

He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

2011-01-01

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells

PubMed Central

2014-01-01

Background The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B). Results Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C. Conclusion AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways. PMID:25201625

Computerized Testing Software for Assessing Interference Suppression in Children and Adults: The Bivalent Shape Task (BST)

PubMed Central

Mueller, Shane T.; Esposito, Alena G.

2015-01-01

We describe the Bivalent Shape Task (BST), software using the Psychology Experiment Building Language (PEBL), for testing of cognitive interference and the ability to suppress interference. The test is available via the GNU Public License, Version 3 (GPLv3), is freely modifiable, and has been tested on both children and adults and found to provide a simple and fast non-verbal measure of cognitive interference and suppression that requires no reading. PMID:26702358

Adaptive arrays for satellite communications

NASA Technical Reports Server (NTRS)

Gupta, I. J.; Ksienski, A. A.

1984-01-01

The suppression of interfering signals in a satellite communication system was studied. Adaptive arrays are used to suppress interference at the reception site. It is required that the interference be suppressed to very low levels and a modified adaptive circuit is used which accomplishes the desired objective. Techniques for the modification of the transmit patterns to minimize interference with neighboring communication links are explored.

RNA virus interference via CRISPR/Cas13a system in plants.

PubMed

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

2018-01-04

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

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

Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide

Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easiermore » to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.« less

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

PubMed Central

Shukla, Jayendra Nath; Palli, Subba Reddy

2014-01-01

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes. PMID:24056158

Cellular microRNA-miR-548g-3p modulates the replication of dengue virus.

PubMed

Wen, Weitao; He, Zhenjian; Jing, Qinlong; Hu, Yiwen; Lin, Cuiji; Zhou, Rui; Wang, Xiaoqun; Su, Yangfan; Yuan, Jiehao; Chen, Zhenxin; Yuan, Jie; Wu, Jueheng; Li, Jun; Zhu, Xun; Li, Mengfeng

2015-06-01

It has been well recognized that microRNA plays a role in the host-pathogen interaction network. The significance of microRNA in the regulation of dengue virus (DENV) replication, however, remains unknown. The objective of our study was to determine the biological function of miR-548g-3p in modulating the replication of dengue virus. Here we report that employment of a microRNA target search algorithm to analyze the 5' untranslated region (5'UTR) consensus sequences of DENV (DENV serotypes 1-4) led to a discovery that miR-548g-3p directly targets the stem loop A promoter element within the 5'UTR, a region essential for DENV replication. Real-time PCR was used to measure the expression levels of miR-548g-3p under DENV infection. We performed overexpression and inhibition assays to test the role of miR-548g-3p on DENV replication. The protein and mRNA levels of interferon were measured by ELISA and real-time PCR respectively. We found that overexpression of miR-548g-3p suppressed multiplication of DENV 1, 2, 3 and 4, and that miR-548g-3p was also found to interfere with DENV translation, thereby suppressing the expression of viral proteins. Our results suggest that miR-548g-3p directly regulates DENV replication and warrant further study to investigate the feasibility of microRNA-based anti-DENV approaches. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Below-baseline suppression of competitors during interference resolution by younger but not older adults.

PubMed

Healey, M Karl; Ngo, K W Joan; Hasher, Lynn

2014-01-01

Resolving interference from competing memories is a critical factor in efficient memory retrieval, and several accounts of cognitive aging suggest that difficulty resolving interference may underlie memory deficits such as those seen in the elderly. Although many researchers have suggested that the ability to suppress competitors is a key factor in resolving interference, the evidence supporting this claim has been the subject of debate. Here, we present a new paradigm and results demonstrating that for younger adults, a single retrieval attempt is sufficient to suppress competitors to below-baseline levels of accessibility even though the competitors are never explicitly presented. The extent to which individual younger adults suppressed competitors predicted their performance on a memory span task. In a second experiment, older adults showed no evidence of suppression, which supports the theory that older adults' memory deficits are related to impaired suppression.

Ethical Perspectives on RNA Interference Therapeutics

PubMed Central

Ebbesen, Mette; Jensen, Thomas G.; Andersen, Svend; Pedersen, Finn Skou

2008-01-01

RNA interference is a mechanism for controlling normal gene expression which has recently begun to be employed as a potential therapeutic agent for a wide range of disorders, including cancer, infectious diseases and metabolic disorders. Clinical trials with RNA interference have begun. However, challenges such as off-target effects, toxicity and safe delivery methods have to be overcome before RNA interference can be considered as a conventional drug. So, if RNA interference is to be used therapeutically, we should perform a risk-benefit analysis. It is ethically relevant to perform a risk-benefit analysis since ethical obligations about not inflicting harm and promoting good are generally accepted. But the ethical issues in RNA interference therapeutics not only include a risk-benefit analysis, but also considerations about respecting the autonomy of the patient and considerations about justice with regard to the inclusion criteria for participation in clinical trials and health care allocation. RNA interference is considered a new and promising therapeutic approach, but the ethical issues of this method have not been greatly discussed, so this article analyses these issues using the bioethical theory of principles of the American bioethicists, Tom L. Beauchamp and James F. Childress. PMID:18612370

Splice isoform-specific suppression of the CaV2.1 variant underlying Spinocerebellar ataxia type 6

PubMed Central

Tsou, Wei-Ling; Soong, Bing-Wen; Paulson, Henry L.; Rodríguez-Lebrón, Edgardo

2011-01-01

Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the CaV2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a CaV2.1 mini-gene reporter system, we found that pathogenic CAG expansions in CaV2.1 enhance splicing activity at the 3′end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding CaV2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding CaV2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding CaV2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases. PMID:21550405

Splice isoform-specific suppression of the Cav2.1 variant underlying spinocerebellar ataxia type 6.

PubMed

Tsou, Wei-Ling; Soong, Bing-Wen; Paulson, Henry L; Rodríguez-Lebrón, Edgardo

2011-09-01

Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Ca(V)2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a Ca(V)2.1 mini-gene reporter system, we found that pathogenic CAG expansions in Ca(V)2.1 enhance splicing activity at the 3'end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding Ca(V)2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding Ca(V)2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding Ca(V)2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

PubMed

Killiny, Nabil; Hajeri, Subhas; Tiwari, Siddharth; Gowda, Siddarame; Stelinski, Lukasz L

2014-01-01

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.

Double-Stranded RNA Uptake through Topical Application, Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in Diaphorina citri

PubMed Central

Killiny, Nabil; Hajeri, Subhas; Tiwari, Siddharth; Gowda, Siddarame; Stelinski, Lukasz L.

2014-01-01

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development. PMID:25330026

MafA is a Key Molecule in Glucose and Energy Balance in the Central Nervous System and Peripheral Organs

PubMed Central

Tsuchiya, Mariko; Tsuchiya, Ken; Yasuda, Kazuki; Fujita, Mikiko; Takinishi, Akira; Furukawa, Maiko; Nitta, Kosaku; Maeda, Atsushi

2011-01-01

MafA is a strong transactivator of insulin in pancreatic β cells. Elucidating the profile of MafA action in organs other than the pancreas is essential. We established an mRNA interference technique that modifies the level of target mRNAs in mice in vivo. After rapidly injecting MafA-siRNA, the resulting changes in the gene profile were analyzed using a microarray system. Significant suppression of the MafA mRNA levels was observed in the pancreas, liver, adipose tissue, and brain of siRNA-injected mice. As we reported previously, the down-regulation of insulin mRNA and adipocytokines was observed in the pancreas, and MafA siRNA caused alterations in the expressions of genes related to lipid metabolism and cell growth in the liver, and the attenuation of cell differentiation in cultured adipocytes. In addition to the effects on these organs, MafA expression was immunohistochemically detected in the brain in our preliminary data, and the expression level in siRNA-treated mice was significantly suppressed. The expressions of the affected genes were distinct, including growth hormone, vasopressin, hypocretin, and pro-melanin-concentrating hormone, were almost completely down-regulated (to ~1/100). These results suggested that MafA is likely involved in the regulation of hormonal systems related to glucose metabolism, and MafA is likely positioned near the beginning of the cascade or may influence the expressions of the above-mentioned genes in coordination with other factors in brain tissue. Taken together, the findings in this study suggested that MafA functions as a transcription factor with distinct activities in each organ and is cross-linked in several organs. PMID:23675216

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

PubMed Central

Kaake, Robyn; Dawson, Anthony R.; Matheos, Dina; Nagashima, Kunio; Sitlani, Parth; Patterson, Kurt; Chang, Ivan; Huang, Lan; Sandmeyer, Suzanne

2015-01-01

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs. PMID:26421679

[Expression analysis of a transformer gene in Daphnia pulex after RNAi].

PubMed

Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L

2016-01-01

In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

PubMed Central

Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J

2010-01-01

Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme’s first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference. PMID:20132829

RNA interference of pheromone biosynthesis-activating neuropeptide receptor suppresses mating behavior by inhibiting sex pheromone production in Plutella xylostella (L.).

PubMed

Lee, Dae-Weon; Shrestha, Sony; Kim, A Young; Park, Seok Joo; Yang, Chang Yeol; Kim, Yonggyun; Koh, Young Ho

2011-04-01

Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms.

PubMed

Bitko, Vira; Musiyenko, Alla; Bayfield, Mark A; Maraia, Richard J; Barik, Sailen

2008-08-01

The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

PubMed

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

PubMed Central

Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

2016-01-01

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

Skin tumor formation in human papillomavirus 8 transgenic mice is associated with a deregulation of oncogenic miRNAs and their tumor suppressive targets.

PubMed

Hufbauer, Martin; Lazić, Daliborka; Reinartz, Markus; Akgül, Baki; Pfister, Herbert; Weissenborn, Sönke Jan

2011-10-01

Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks. Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice. Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining. Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated. This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

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

Nitschke, Matthias; Korte, Thomas; Tielesch, Claudia

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggestmore » that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.« less

RNAi or overexpression: Alternative therapies for Spinocerebellar Ataxia Type 1

PubMed Central

Keiser, Megan S.; Geoghegan, James C.; Boudreau, Ryan L.; Lennox, Kim A.; Davidson, Beverly L.

2014-01-01

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant late onset neurodegenerative disease caused by an expanded polyglutamine tract in ataxin-1. Here, we compared the protective effects of overexpressing ataxin-1-like using recombinant AAVs, or reducing expression of mutant ataxin-1 using virally delivered RNA interference (RNAi), in a transgenic mouse model of SCA1. For the latter, we used an artificial microRNA (miR) design that optimizes potency, efficacy and safety to suppress ataxin-1 expression (miS1). Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mice cerebella resulted in widespread cerebellar Purkinje cell transduction and improved behavioral and histological phenotypes. Our data indicate the utility of either approach as a possible therapy for SCA1 patients. PMID:23583610

DLK1 as a potential target against cancer stem/progenitor cells of hepatocellular carcinoma.

PubMed

Xu, Xiao; Liu, Rui-Fang; Zhang, Xin; Huang, Li-Yu; Chen, Fei; Fei, Qian-Lan; Han, Ze-Guang

2012-03-01

Delta-like 1 homolog (DLK1; Drosophila) is a hepatic stem/progenitor cell marker in fetal livers that plays a vital role in oncogenesis of hepatocellular carcinoma (HCC). The aim of this study is to investigate whether DLK1 could serve as a potential therapeutic target against cancer stem/progenitor cells of HCC. DLK1(+) and DLK1(-) cells were sorted by fluorescence-activated cell sorting and magnetic-activated cell sorting, respectively, and then were evaluated by flow cytometry. The biological behaviors of these isolated cells and those with DLK1 knockdown were assessed by growth curve, colony formation assay, spheroid colony formation, chemoresistance, and in vivo tumorigenicity. Adenovirus-mediated RNA interference was used to knockdown the endogenous DLK1. We found that DLK1(+) population was less than 10% in almost all 17 HCC cell lines examined. DLK1(+) HCC cells showed stronger ability of chemoresistance, colony formation, spheroid colony formation, and in vivo tumorigenicity compared with DLK1(-) cells. The DLK1(+) HCC cells could generate the progeny without DLK1 expression. Furthermore, DLK1 knockdown could suppress the ability of proliferation, colony formation, spheroid colony formation, and in vivo tumorigenicity of Hep3B and Huh-7 HCC cells. Our data suggested that DLK1(+) HCC cells have characteristics similar to those of cancer stem/progenitor cells. RNA interference against DLK1 can suppress the malignant behaviors of HCC cells, possibly through directly disrupting cancer stem/progenitor cells, which suggested that DLK1 could be a potential therapeutic target against the HCC stem/progenitor cells.

Genetic manipulation of isoprene emissions in poplar plants remodels the chloroplast proteome.

PubMed

Velikova, Violeta; Ghirardo, Andrea; Vanzo, Elisa; Merl, Juliane; Hauck, Stefanie M; Schnitzler, Jörg-Peter

2014-04-04

Biogenic isoprene (2-methyl-1,3-butadiene) improves the integrity and functionality of thylakoid membranes and scavenges reactive oxygen species (ROS) in plant tissue under stress conditions. On the basis of available physiological studies, we hypothesized that the suppression of isoprene production in the poplar plant by genetic engineering would cause changes in the chloroplast protein pattern, which in turn would compensate for changes in chloroplast functionality and overall plant performance under abiotic stress. To test this hypothesis, we used a stable isotope-coded protein-labeling technique in conjunction with polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry. We analyzed quantitative and qualitative changes in the chloroplast proteome of isoprene-emitting and non isoprene-emitting poplars. Here we demonstrate that suppression of isoprene synthase by RNA interference resulted in decreased levels of chloroplast proteins involved in photosynthesis and increased levels of histones, ribosomal proteins, and proteins related to metabolism. Overall, our results show that the absence of isoprene triggers a rearrangement of the chloroplast protein profile to minimize the negative stress effects resulting from the absence of isoprene. The present data strongly support the idea that isoprene improves/stabilizes thylakoid membrane structure and interferes with the production of ROS.

Reprogramming the phenylpropanoid metabolism in seeds of oilseed rape by suppressing the orthologs of reduced epidermal fluorescence1.

PubMed

Mittasch, Juliane; Böttcher, Christoph; Frolov, Andrej; Strack, Dieter; Milkowski, Carsten

2013-04-01

As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene reduced epidermal fluorescence1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression.

Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs of REDUCED EPIDERMAL FLUORESCENCE11[W

PubMed Central

Mittasch, Juliane; Böttcher, Christoph; Frolov, Andrej; Strack, Dieter; Milkowski, Carsten

2013-01-01

As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression. PMID:23424250

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

PubMed Central

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

2014-01-01

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

Metallothioneins regulate the adipogenic differentiation of 3T3-L1 cells via the insulin signaling pathway

PubMed Central

Toriuchi, Yuriko; Aki, Yuka; Mizuno, Yuto; Kawakami, Takashige; Nakaya, Tomoko; Sato, Masao; Suzuki, Shinya

2017-01-01

Knockout of metallothionein (MT) genes contributes to a heavier body weight in early life and the potential to become obese through the intake of a high fat diet (HFD) in mice. It has thus been suggested that MT genes regulate the formation of adipose tissue, which would become the base for later HFD-induced obesity. We evaluated the fat pads of mice during the lactation stage. The fat mass and adipocyte size of MT1 and MT2 knockout mice were greater than those of wild type mice. Next, we assayed the ability of small interfering RNA (siRNA) to silence MT genes in the 3T3-L1 cell line. The expressions of MT1 and MT2 genes were transiently upregulated during adipocyte differentiation, and the siRNA pretreatment led to the suppression of the expression of both MT mRNAs and proteins. The MT siRNA promoted lipid accumulation in adipocytes and caused proliferation of post-confluent preadipocytes; these effects were suppressed by an inhibitor of phosphatidylinositol 3-kinase (LY294002). In addition, MT siRNA promoted insulin-stimulated phosphorylation of Akt, a downstream kinase of the insulin signaling pathway. Enhanced lipid accumulation in 3T3-L1 cells resulting from MT-gene silencing was inhibited by pretreatment with an antioxidant, N-acetylcysteine, used as a substitute for antioxidant protein MTs. These results suggest that interference in MT expression enhanced the activation of the insulin signaling pathway, resulting in higher lipid accumulation in 3T3-L1 adipocytes. PMID:28426713

Persistent interferon transgene expression by RNA interference-mediated silencing of interferon receptors.

PubMed

Takahashi, Yuki; Vikman, Elin; Nishikawa, Makiya; Ando, Mitsuru; Watanabe, Yoshihiko; Takakura, Yoshinobu

2010-09-01

The in vivo half-life of interferons (IFNs) is very short, and its extension would produce a better therapeutic outcome in IFN-based therapy. Delivery of IFN genes is one solution for providing a sustained supply. IFNs have a variety of functions, including the suppression of transgene expression, through interaction with IFN receptors (IFNRs). This suppression could prevent IFNs from being expressed from vectors delivered. Silencing the expression of IFNAR and IFNGR, the receptors for type I and II IFNs, respectively, in cells expressing IFNs may prolong transgene expression of IFNs. Mouse melanoma B16-BL6 cells or mouse liver were selected as a site expressing IFNs (not a target for IFN gene therapy) and IFN-expressing plasmid DNA was delivered with or without small interfering RNA (siRNA) targeting IFNRs. Transfection of B16-BL6 cells with siRNA targeting IFNAR1 subunit (IFNAR1) resulted in the reduced expression of IFNAR on the cell surface. This silencing significantly increased the IFN-beta production in cells that were transfected with IFN-beta-expressing plasmid DNA. Similar results were obtained with the combination of IFN-gamma and IFNGR. Co-injection of IFN-beta-expressing plasmid DNA with siRNA targeting IFNAR1 into mice resulted in sustained plasma concentration of IFN-beta. These results provide experimental evidence that the RNAi-mediated silencing of IFNRs in cells expressing IFN, such as hepatocytes, is an effective approach for improving transgene expression of IFNs when their therapeutic target comprises cells other than those expressing IFNs.

Inhibition of alpha interferon (IFN-α)-induced microRNA-122 negatively affects the anti-hepatitis B virus efficiency of IFN-α.

PubMed

Hao, Junli; Jin, Wensong; Li, Xinghui; Wang, Saifeng; Zhang, Xiaojun; Fan, Hongxia; Li, Changfei; Chen, Lizhao; Gao, Bin; Liu, Guangze; Meng, Songdong

2013-01-01

Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3'-untranslated region (3'-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy.

Inhibition of Alpha Interferon (IFN-α)-Induced MicroRNA-122 Negatively Affects the Anti-Hepatitis B Virus Efficiency of IFN-α

PubMed Central

Hao, Junli; Jin, Wensong; Li, Xinghui; Wang, Saifeng; Zhang, Xiaojun; Fan, Hongxia; Li, Changfei; Chen, Lizhao; Gao, Bin

2013-01-01

Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3′-untranslated region (3′-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy. PMID:23055569

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

PubMed Central

He, Hongbin; Ding, Fangrong; Yang, Hongjun; Cheng, Lei; Liu, Wenhao; Zhong, Jifeng; Dai, Yunping; Li, Guangpeng; He, Chengqiang; Yu, Li; Li, Jianbin

2012-01-01

Background Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. Principal Finding Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. Conclusion RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV. PMID:22905125

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females.

PubMed

Shukla, Jayendra Nath; Palli, Subba Reddy

2013-12-01

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes. Copyright © 2013 Elsevier Ltd. All rights reserved.

The asparagine residue in the FRNK box of potyviral helper-component protease is critical for its small RNA binding and subcellular localization.

PubMed

Sahana, Nandita; Kaur, Harpreet; Jain, R K; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2014-05-01

The multifunctional potyviral helper-component protease (HcPro) contains variable regions with some functionally conserved domains, such as the FRNK box. Natural variants occur at the FRNK box, a conserved central domain, known for its role in RNA binding and RNAi suppression activities, although no dominant natural variants for the N(182) residue are known to occur. Here, a mutant at HcPro(N182L) was developed to investigate its role in natural populations. Using in vitro studies, we found an increase in the small RNA (sRNA) binding potential of HcPro(N182L) without affecting its protein-protein interaction properties, suggesting that the presence of N(182) is critical to maintain threshold levels of sRNAs, but does not interfere in the self-interaction of HcPro. Furthermore, we found that expression of HcPro(N182L) in Nicotiana benthamiana affected plant growth. Transient expression of HcPro(N182L) induced reporter gene expression in 16c GFP transgenic plants more than HcPro did, suggesting that replacement of asparagine in the FRNK box favours RNA silencing suppression. HcPro was found to be distributed in the nucleus and cytoplasm, whereas HcPro(N182L) was observed only in cytoplasmic inclusion bodies in N. benthamiana leaves, when fused to a GFP tag and expressed by agro-infiltration, suggesting mutation favours oligomerization of HcPro. These findings suggest that amino acid N(182) of the conserved FRNK box may regulate RNA silencing mechanisms, and is required for maintenance of the subcellular localization of the protein for its multi-functionality. Hence, the N(182) residue of the FRNK box seems to be indispensable for potyvirus infection during evolution.

Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes.

PubMed

Toyota, Naoji; Fujitsuka, Chiaki; Ishibashi, Goushi; S Yoshida, Lucia; Takano-Ohmuro, Hiromi

2016-01-01

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55γ as a Stress-Sensitive Inhibitor of c-SRC

PubMed Central

Eichhorn, Pieter J. A; Creyghton, Menno P; Wilhelmsen, Kevin; van Dam, Hans; Bernards, René

2007-01-01

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH2-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ. PMID:18069897

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

PubMed

Yoon, Dong Suk; Choi, Yoorim; Jang, Yeonsue; Lee, Moses; Choi, Woo Jin; Kim, Sung-Hwan; Lee, Jin Woo

2014-12-01

SOX2 is crucial for the maintenance of the self-renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)-derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self-renewal and differentiation capacities of BM-MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM-MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM-MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA-mediated acetylation and ubiquitination of SOX2 in BM-MSCs. SIRT1 activation by resveratrol enhanced the colony-forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose-dependent manner. However, the enhancement of self-renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1-SOX2 axis plays an important role in maintaining the self-renewal capability and multipotency of BM-MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post-translational modification in BM-MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1-mediated deacetylation contributes to maintaining SOX2 protein in the nucleus. © 2014 AlphaMed Press.

Upregulation of human DNA binding protein A (dbpA) in gastric cancer cells.

PubMed

Wang, Guo-rong; Zheng, Yan; Che, Xiang-ming; Wang, Xin-yang; Zhao, Jia-hui; Wu, Kai-jie; Zeng, Jin; Pan, Chen-en; He, Da-lin

2009-10-01

To determine the effect of human DNA binding protein (dbpA) on the biology of gastric cancer cells. DbpA expression was analyzed by Western blot analysis and immunofluorescence staining in gastric cancer tissues and cell lines. A dbpA-specific small interference (si) RNA was designed and synthesized. Suppressive effect of siRNA on dbpA expression was assessed by real-time RT-PCR. Transwell migration and colony formation assays were used to assess the inhibitory effects of dbpA siRNA on cell invasion and tumorigenesis in vitro. Drug-sensitivity was evaluated using a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of dbpA was upregulated in gastric cancer tissues and cell lines as compared to adjacent normal tissues or gastric epithelial cells. siRNA treatment successfully silenced dbpA expression. Silencing of dbpA increased expression of E-cadherin, decreased expression of adenomatous polyposis coli (APC), beta-catenin and cyclin D1, but had no effect on expression of NF-kappaB. Silencing of dbpA also suppressed cell invasion and colony formation of SGC7901 cells, and enhanced their chemosensitivity to 5-fluorouracil. DbpA plays an important role in the pathogenesis and development of gastric cancer, and the process involves E-cadherin, APC, beta-catenin and cyclin D1. Silencing of dbpA might be a novel therapeutic strategy for increasing chemosensitivity to 5-fluorouracil in gastric cancer.

Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.

PubMed

Wang, Yun-Liang; Dong, Feng-Lin; Yang, Jian; Li, Zhi; Zhi, Qiao-Ming; Zhao, Xin; Yang, Yong; Li, De-Chun; Shen, Xiao-Chun; Zhou, Jin

2015-01-01

Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1

PubMed Central

Barbie, David A.; Tamayo, Pablo; Boehm, Jesse S.; Kim, So Young; Moody, Susan E.; Dunn, Ian F.; Schinzel, Anna C.; Sandy, Peter; Meylan, Etienne; Scholl, Claudia; Fröhling, Stefan; Chan, Edmond M.; Sos, Martin L.; Michel, Kathrin; Mermel, Craig; Silver, Serena J.; Weir, Barbara A.; Reiling, Jan H.; Sheng, Qing; Gupta, Piyush B.; Wadlow, Raymond C.; Le, Hanh; Hoersch, Sebastian; Wittner, Ben S.; Ramaswamy, Sridhar; Livingston, David M.; Sabatini, David M.; Meyerson, Matthew; Thomas, Roman K.; Lander, Eric S.; Mesirov, Jill P.; Root, David E.; Gilliland, D. Gary; Jacks, Tyler; Hahn, William C.

2009-01-01

The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele1,2. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IκB kinase, TBK1, was selectively essential in cells that harbor mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-κB anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 and NF-κB signaling as essential in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer. PMID:19847166

Renewing the Assault on mRNA

PubMed Central

McCAIN, JACK

2004-01-01

Mammalian cells dislike double-stranded RNA. They interpret it as a sign of an intruder, and they can unleash a recently discovered defensive mechanism to deal with the problem – they chop the invader into little pieces and use the remnants, called small interfering RNA, to identify and destroy the invader and its progeny. This process, known as RNA interference, may lend itself to new treatments for a wide range of diseases. RNA interference, however, resembles two therapies studied during the 1990s, antisense and ribozymes, in that the gene-silencing target is messenger RNA (mRNA). Is RNA interference really the Next Big Thing – or just a variation on an older but still intriguing theme? PMID:23372488

RNA interference targeting E637K mutation rescues hERG channel currents and restores its kinetic properties.

PubMed

Lu, Xiaoli; Yang, Xi; Huang, Xiaoyan; Huang, Chen; Sun, Huan Huan; Jin, Lihua; Xu, Weifeng; Mao, Haiyan; Guo, Junming; Zhou, Jianqing; Lian, Jiangfang

2013-01-01

Long QT syndrome (LQTS) is a monogenic proarrhythmic disorder that predisposes affected individuals to sudden death from tachyarrhythmia. As an inherited disease, LQTS cannot be completely cured by conventional treatment modalities. Individualized gene therapy is a promising therapeutic approach. The purpose of this study was to investigate the role of small interference RNA (siRNA) on expression of E637K-hERG (human ether-a-go-go-related gene) mutant and whether it can be used to rescue the mutant's dominant-negative suppressive effects on hERG protein channel function. Western blot was performed to select the most sensitive siRNAs to target E637K-hERG mutant knockdown. Confocal laser scanning microscope was performed to monitor cellular localization of wild-type (WT)-hERG and E637K-hERG with or without siRNA. Patch-clamp technique was used to assess the effect of siRNA on the electrophysiologic characteristics of the rapidly activating delayed rectifier K(+) current I(Kr) of the hERG protein channel. siRNA led to a significant decrease in the level of E637K-hERG protein but did not affect the level of WT-hERG protein. WT-hERG localization in cells coexpressing E637K-hERG mutant was restored to the membrane by siRNA. The siRNA-mediated inhibition of E637K-hERG mutant restored the maximum current and tail current amplitudes. Furthermore, siRNA treatment rescued the kinetic properties of WT/E637K-hERG protein channel to a level comparable to that of WT-hERG protein channel. Our findings illustrated that siRNA can effectively inhibit E637K-hERG protein expression and rescue the dominant-negative effect of this mutation by restoring the kinetic properties of hERG protein channel. It has potential clinical implications with regard to the possibility of using siRNA in the treatment of LQTS. Copyright © 2013 Heart Rhythm Society. All rights reserved.

RNAi of COL1A1 in mesenchymal progenitor cells.

PubMed

Millington-Ward, Sophia; McMahon, Helena P; Allen, Danny; Tuohy, Gearóid; Kiang, Anna-Sophia; Palfi, Arpad; Kenna, Paul F; Humphries, Peter; Farrar, G Jane

2004-10-01

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

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

PubMed

Whitten, Miranda; Dyson, Paul

2017-03-01

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

Roles of Heparan Sulfate Sulfation in Dentinogenesis*

PubMed Central

Hayano, Satoru; Kurosaka, Hiroshi; Yanagita, Takeshi; Kalus, Ina; Milz, Fabian; Ishihara, Yoshihito; Islam, Md. Nurul; Kawanabe, Noriaki; Saito, Masahiro; Kamioka, Hiroshi; Adachi, Taiji; Dierks, Thomas; Yamashiro, Takashi

2012-01-01

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix. PMID:22351753

Chronic Cardiac-Targeted RNA Interference for the Treatment of Heart Failure Restores Cardiac Function and Reduces Pathological Hypertrophy

PubMed Central

Suckau, Lennart; Fechner, Henry; Chemaly, Elie; Krohn, Stefanie; Hadri, Lahouaria; Kockskämper, Jens; Westermann, Dirk; Bisping, Egbert; Ly, Hung; Wang, Xiaomin; Kawase, Yoshiaki; Chen, Jiqiu; Liang, Lifan; Sipo, Isaac; Vetter, Roland; Weger, Stefan; Kurreck, Jens; Erdmann, Volker; Tschope, Carsten; Pieske, Burkert; Lebeche, Djamel; Schultheiss, Heinz-Peter; Hajjar, Roger J.; Poller, Wolfgang Ch.

2009-01-01

Background RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. We report on targeted RNAi for the treatment of heart failure (HF), an important disorder in humans resulting from multiple etiologies. Successful treatment of HF is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban (PLB), a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy employs regulatory RNAs to achieve its effect. Methods and Results We describe structural requirements to obtain high RNAi activity from adenoviral (AdV) and adeno-associated virus (AAV9) vectors and show that an AdV short hairpin RNA vector (AdV-shRNA) silenced PLB in cardiomyocytes (NRCMs) and improved hemodynamics in HF rats 1 month after aortic root injection. For simplified long-term therapy we developed a dimeric cardiotropic AAV vector (rAAV9-shPLB) delivering RNAi activity to the heart via intravenous injection. Cardiac PLB protein was reduced to 25% and SERCA2a suppression in the HF groups was rescued. In contrast to traditional vectors rAAV9 shows high affinity for myocardium, but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (LVEDP, dp/dtmin, Tau) and systolic (fractional shortening) functional parameters to normal range. The massive cardiac dilation was normalized and the cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis significantly reduced. Importantly, there was no evidence of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusion Our data show, for the first time, high efficacy of an RNAi therapeutic strategy in a cardiac disease. PMID:19237664

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

Fan, Qi; Wang, Xuedi; Zhang, Hanguang

Highlights: Black-Right-Pointing-Pointer Cat S is highly expressed in HCC cells with high metastatic potential. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits growth and invasion of HCC cells. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits HCC-associated angiogenesis. Black-Right-Pointing-Pointer Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCCmore » cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.« less

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

Interference with endogenous EZH2 reverses the chemotherapy drug resistance in cervical cancer cells partly by up-regulating Dicer expression.

PubMed

Cai, Liqiong; Wang, Zehua; Liu, Denghua

2016-05-01

Cervical cancer is one of the most common female malignancies in the world, and chemotherapeutic drug resistance is a major obstacle to cancer therapy. Enhancer of zeste homolog 2 (EZH2) is an enzymatic subunit of polycomb repressive complex 2 (PRC2) and catalyzes the repressive histone H3 lysine 27 trimethylation (H3K27me3). However, the role of EZH2 on the chemotherapy drug resistance in cervical cancers remains unclear. In the present study, the cervical carcinoma specimens and paired normal tissue specimens were obtained and the expression of EZH2 was detected by western blotting. The results showed that high levels of EZH2 were detected in cervical carcinoma tissues, compared with paired control tissues (**p < 0.01). Next, three pairs of shRNA specific to EZH2 were designed and used to interfere with endogenous EZH2 expression. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays following treatment with various concentrations of cisplatin in HeLa and HeLa/DDP cells. The MTT assay results showed that knockdown of EZH2 in HeLa/DDP cells caused a 2.29- or 1.83-fold decrease in the cisplatin IC50 values (for shRNA1-EZH2, 34.88 vs. 15.21 μg/mL; p < 0.01; for shRNA3-EZH2, 34.88 vs. 19.09 μg/mL; p < 0.01). The EZH2 activity was also suppressed by 3-deazaneplanocin A (DZNep), EZH2 inhibitor, and the results demonstrated that, meanwhile, DZNep potently inhibited cell viability of HeLa/DDP cells, partly by suppression the levels of EZH2 and H3K27me3, but not H3K27me2, which was detected by western blotting analysis. Moreover, cell migration assay results showed that knockdown of EZH2 decreased cell metastasis of cervical cancer cells. Furthermore, cell cycle was detected by fluorescence-activated cell sorting (FACS) assay and the results demonstrated that interference with EZH2 expression increased the percentage of cells at G0/G1 phase and the HeLa/DDP cells were blocked at G0/G1 phase. Interestingly, western blotting results revealed that higher expression of EZH2 was related with lower level of Dicer in HeLa/DDP cells. Finally, in vivo tumorigenicity experiments results demonstrated that interference with endogenous EZH2 by shRNA specific to EZH2 or inhibition EZH2 by DZNep could significantly increase antitumor effects in nude mice. Thus, inhibiting the levels of endogenous EZH2 effectively reversed the cisplatin resistance and increased the cisplatin sensitivity in cisplatin-resistant HeLa/DDP cells. EZH2 might be a potential target for treating chemotherapeutic drug-resistant cervical cancers.

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.

PubMed

Smith, Justin D; Suresh, Sundari; Schlecht, Ulrich; Wu, Manhong; Wagih, Omar; Peltz, Gary; Davis, Ronald W; Steinmetz, Lars M; Parts, Leopold; St Onge, Robert P

2016-03-08

Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

RNA-protein interactions in plant disease: hackers at the dinner table.

PubMed

Spanu, Pietro D

2015-09-01

Plants are the source of most of our food, whether directly or as feed for the animals we eat. Our dinner table is a trophic level we share with the microbes that also feed on the primary photosynthetic producers. Microbes that enter into close interactions with plants need to evade or suppress detection and host immunity to access nutrients. They do this by deploying molecular tools - effectors - which target host processes. The mode of action of effector proteins in these events is varied and complex. Recent data from diverse systems indicate that RNA-interacting proteins and RNA itself are delivered by eukaryotic microbes, such as fungi and oomycetes, to host plants and contribute to the establishment of successful interactions. This is evidence that pathogenic microbes can interfere with the host software. We are beginning to see that pathogenic microbes are capable of hacking into the plants' immunity programs. © 2015 The Author. New Phytologist © 2015 New Phytologist Trust.

Reduced STMN1 expression induced by RNA interference inhibits the bioactivity of pancreatic cancer cell line Panc-1.

PubMed

Li, J; Hu, G H; Kong, F J; Wu, K M; He, B; Song, K; Sun, W J

2014-01-01

Increased expression of STMN1 has been observed in many tumor forms, but its expression and potential biological role in pancreatic cancer is still unknown. In this study, we demonstrated that STMN1 was expressed to a large extent in pancreatic cancer tissues and cell lines as compared to normal pancreatic tissues. Suppression of STMN1 expression via transfection with STMN1-specific siRNA could not only significantly inhibit the proliferation, migration and invasion ability of Panc-1 cells, but also enhance the apoptosis of Panc-1 cells. In addition, downregulation of STMN1 obviously enhanced the acetylation level of α-tubulin. All these results indicated that STMN1 plays an important role in pancreatic cancer development, and might serve as a potential therapeutic target for pancreatic cancer.

Stimuli-Responsive Nano-Carrier for Co-delivery of MiR-31 and Doxorubicin to Suppress High MtEF4 Cancer.

PubMed

Wang, Fang; Zhang, Lingyun; Bai, Xiufeng; Cao, Xintao; Jiao, Xiangyu; Huang, Yan; Li, Yansheng; Qin, Yan; Wen, Yongqiang

2018-06-13

Gene interference-based therapeutics represents a fascinating challenge and shows enormous potential for cancer treatment, in which microRNA is used to correct abnormal gene. Based on the above, we introduced microRNA-31 to bind to 3' untranslated region of mtEF4, resulting in the downregulation of its messenger RNA and protein to trigger cancer cells apoptosis through mitochondria-related pathway. To achieve better therapeutic effect, a mesoporous silica nanoparticles-based controlled nanoplatform had been developed. This system was fabricated by conjugation of microRNA-31 onto doxorubicin-loaded mesoporous silica nanoparticles with a PEI/HA coating, and drug release was triggered by acidic environment of tumors. By feat of surface functionalization and tumor-specific conjugation to nanoparticles, our drug delivery system could promote intracellular accumulation of drugs via the active transport at tumor site. More importantly, microRNA-31 not only directly targeted to mtEF4 to promote cells death, but had synergistic effects when used in combination with doxorubicin, and achieved excellent superadditive effects. As such, our research might provide new insights towards detecting high mtEF4 cancer and exploiting highly effective anticancer drugs.

Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system

PubMed Central

Ross, Joseph A.; Ellis, Michael J.; Hossain, Shahan; Haniford, David B.

2013-01-01

Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements. PMID:23510801

Near-Field Noise Source Localization in the Presence of Interference

NASA Astrophysics Data System (ADS)

Liang, Guolong; Han, Bo

In order to suppress the influence of interference sources on the noise source localization in the near field, the near-field broadband source localization in the presence of interference is studied. Oblique projection is constructed with the array measurements and the steering manifold of interference sources, which is used to filter the interference signals out. 2D-MUSIC algorithm is utilized to deal with the data in each frequency, and then the results of each frequency are averaged to achieve the positioning of the broadband noise sources. The simulations show that this method suppresses the interference sources effectively and is capable of locating the source which is in the same direction with the interference source.

The Role of the Left Head of Caudate in Suppressing Irrelevant Words

ERIC Educational Resources Information Center

Ali, Nilufa; Green, David W.; Kherif, Ferath; Devlin, Joseph T.; Price, Cathy J.

2010-01-01

Suppressing irrelevant words is essential to successful speech production and is expected to involve general control mechanisms that reduce interference from task-unrelated processing. To investigate the neural mechanisms that suppress visual word interference, we used fMRI and a Stroop task, using a block design with an event-related analysis.…

Cardiospecific CD36 suppression by lentivirus-mediated RNA interference prevents cardiac hypertrophy and systolic dysfunction in high-fat-diet induced obese mice.

PubMed

Zhang, Yijie; Bao, Mingwei; Dai, Mingyan; Wang, Xin; He, Wenbo; Tan, Tuantuan; Lin, Dandan; Wang, Wei; Wen, Ying; Zhang, Rui

2015-06-03

Fatty acid (FA) catabolism abnormality has been proved to play an important role in obesity-related cardiomyopathy. We hypothesized that cardiospecific suppression of CD36, the predominant membrane FA transporter, would protect against obesity-related cardiomyopathy. Four-wk-old male C57BL/6 J mice were fed with either high-fat-diet (HFD) or control-normal-diet for 2 wk. Then they were subjected to intramyocardial injection with recombinant lentiviral vectors containing short hairpin RNAs to selectively downregulate the expression of either cardiac CD36 or irrelevant gene by RNA interference. After a 10-wk continuation of the diet, biochemical, functional, morphological, histological, metabolic and molecular profiles were assessed. HFD administration elicited obesity, cardiac hypertrophy and systolic dysfunction accompanied with elevated serum levels of blood urea nitrogen (BUN), creatinine, fasting serum glucose (FSG), total cholesterol (TC) and triglyceride. Additionally, HFD consumption promoted lipid accumulation and reactive oxygen species (ROS) generation in the cardiomyocytes. Cardiospecific CD36 inhibition protected against HFD induced cardiac remodeling by decreasing heart/body weight ratio, increasing left ventricular (LV) ejection fraction and fractional shortening as well as normalizing LV diameter, without influencing body weight gain. Inhibition of cardiac CD36 also mitigated obesity induced alteration in BUN, creatinine and triglyceride, but had no effect on FSG or TC. Moreover, cardiospecific CD36 deficiency corrected myocardial lipid overaccumulation and intracellular ROS overproduction that were induced by HFD feeding. Cardiospecific CD36 inhibition protects against the aggravation of cardiac functional and morphological changes associated with HFD induced obesity. CD36 represents a potential therapeutic target for obesity cardiomyopathy.

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

PubMed Central

Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

1999-01-01

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

Modulation of Human Macrophage Responses to Mycobacterium tuberculosis by Silver Nanoparticles of Different Size and Surface Modification

PubMed Central

Sarkar, Srijata; Leo, Bey Fen; Carranza, Claudia; Chen, Shu; Rivas-Santiago, Cesar; Porter, Alexandra E.; Ryan, Mary P.; Gow, Andrew; Chung, Kian Fan; Tetley, Teresa D.; Zhang, Junfeng (Jim); Georgopoulos, Panos G.; Ohman-Strickland, Pamela A.; Schwander, Stephan

2015-01-01

Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications. PMID:26580078

Modulation of Human Macrophage Responses to Mycobacterium tuberculosis by Silver Nanoparticles of Different Size and Surface Modification.

PubMed

Sarkar, Srijata; Leo, Bey Fen; Carranza, Claudia; Chen, Shu; Rivas-Santiago, Cesar; Porter, Alexandra E; Ryan, Mary P; Gow, Andrew; Chung, Kian Fan; Tetley, Teresa D; Zhang, Junfeng Jim; Georgopoulos, Panos G; Ohman-Strickland, Pamela A; Schwander, Stephan

2015-01-01

Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.

Biochemical analysis of NSs from different tospoviruses.

PubMed

Hedil, Marcio; de Ronde, Dryas; Kormelink, Richard

2017-10-15

Tospoviruses suppress antiviral RNA interference by coding for an RNA silencing suppressor (NSs) protein. Previously, using NSs-containing crude plant and insect cell extracts, the affinity of NSs for double-stranded (ds)RNA molecules was demonstrated by electrophoretic mobility shifts assays (EMSAs). While NSs from tomato spotted wilt virus (TSWV) and groundnut ringspot virus (GRSV) were able to bind small and long dsRNA molecules, the one from tomato yellow ring virus (TYRV), a distinct Asian tospovirus, only bound small dsRNA. Here, using bacterially expressed and purified NSs from GRSV and TYRV, it is shown that they are both able to bind to small and long dsRNA. Binding of siRNAs by NSs revealed two consecutive shifts, i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When NSs of TSWV resistance inducing (RI) and resistance breaking (RB) isolates were analyzed using extracts from infected plants only a major siRNA shift was observed. In contrast, plant extracts containing the respective transiently expressed NSs proteins showed only the lower shift with NSs RI but no shift with NSs RB . The observed affinity for RNA duplexes, as well as the two-stepwise shift pattern, is discussed in light of NSs as a suppressor of silencing and its importance for tospovirus infection. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Design and cloning strategies for constructing shRNA expression vectors

PubMed Central

McIntyre, Glen J; Fanning, Gregory C

2006-01-01

Background Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). Results We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. Conclusion In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process. PMID:16396676

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

Public access defibrillation: suppression of 16.7 Hz interference generated by the power supply of the railway systems.

PubMed

Christov, Ivaylo I; Iliev, Georgi L

2005-03-15

A specific problem using the public access defibrillators (PADs) arises at the railway stations. Some countries as Germany, Austria, Switzerland, Norway and Sweden are using AC railroad net power-supply system with rated 16.7 Hz frequency modulated from 15.69 Hz to 17.36 Hz. The power supply frequency contaminates the electrocardiogram (ECG). It is difficult to be suppressed or eliminated due to the fact that it considerably overlaps the frequency spectra of the ECG. The interference impedes the automated decision of the PADs whether a patient should be (or should not be) shocked. The aim of this study is the suppression of the 16.7 Hz interference generated by the power supply of the railway systems. Software solution using adaptive filtering method was proposed for 16.7 Hz interference suppression. The optimal performance of the filter is achieved, embedding a reference channel in the PADs to record the interference. The method was tested with ECGs from AHA database. The method was tested with patients of normal sinus rhythms, symptoms of tachycardia and ventricular fibrillation. Simulated interference with frequency modulation from 15.69 Hz to 17.36 Hz changing at a rate of 2% per second was added to the ECGs, and then processed by the suggested adaptive filtering. The method totally suppresses the noise with no visible distortions of the original signals. The proposed adaptive filter for noise suppression generated by the power supply of the railway systems has a simple structure requiring a low level of computational resources, but a good reference signal as well.

Identification of germline transcriptional regulatory elements in Aedes aegypti.

PubMed

Akbari, Omar S; Papathanos, Philippos A; Sandler, Jeremy E; Kennedy, Katie; Hay, Bruce A

2014-02-04

The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UD(MEL), and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

Identification of germline transcriptional regulatory elements in Aedes aegypti

NASA Astrophysics Data System (ADS)

Akbari, Omar S.; Papathanos, Philippos A.; Sandler, Jeremy E.; Kennedy, Katie; Hay, Bruce A.

2014-02-01

The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

Detecting changes in the nutritional value and elemental composition of transgenic sorghum grain

NASA Astrophysics Data System (ADS)

Ndimba, R.; Grootboom, A. W.; Mehlo, L.; Mkhonza, N. L.; Kossmann, J.; Barnabas, A. D.; Mtshali, C.; Pineda-Vargas, C.

2015-11-01

We have previously demonstrated that poor digestibility in sorghum can be addressed by using RNA interference (RNAi) to suppress kafirin synthesis. The approach resulted in a twofold improvement in overall protein digestibility levels. In the present study, the effect of this targeted kafirin suppression on other grain quality parameters was investigated. Several significant changes in the proximate composition, amino acid profile and the bulk mineral content were detected. Importantly, the most limiting amino acid, lysine, was significantly increased in the transgenic grains by up to 39%; whilst mineral elements in the bulk, such as sulphur (S) and zinc (Zn) were reduced by up to 15.8% and 21% respectively. Elemental mapping of the grain tissue, using micro-PIXE, demonstrated a significant decrease in Zn (>75%), which was localised to the outer endosperm region, whilst TEM revealed important changes to the protein body morphology of the transgenic grains.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

PubMed

Zhang, Qun; Shu, Fu-Li; Jiang, Yu-Feng; Huang, Xin-En

2015-01-01

In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior to that of single RNA interference, and this could be a contribution for prevention of precancerous condition.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness

PubMed Central

Klocko, Amy L.; Borejsza-Wysocka, Ewa; Brunner, Amy M.; Shevchenko, Olga; Aldwinckle, Herb; Strauss, Steven H.

2016-01-01

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These “double-flowers” had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness. PMID:27500731

[Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

PubMed

Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

2010-10-01

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

Experimental generalized quantum suppression law in Sylvester interferometers

NASA Astrophysics Data System (ADS)

Viggianiello, Niko; Flamini, Fulvio; Innocenti, Luca; Cozzolino, Daniele; Bentivegna, Marco; Spagnolo, Nicolò; Crespi, Andrea; Brod, Daniel J.; Galvão, Ernesto F.; Osellame, Roberto; Sciarrino, Fabio

2018-03-01

Photonic interference is a key quantum resource for optical quantum computation, and in particular for so-called boson sampling devices. In interferometers with certain symmetries, genuine multiphoton quantum interference effectively suppresses certain sets of events, as in the original Hong–Ou–Mandel effect. Recently, it was shown that some classical and semi-classical models could be ruled out by identifying such suppressions in Fourier interferometers. Here we propose a suppression law suitable for random-input experiments in multimode Sylvester interferometers, and verify it experimentally using 4- and 8-mode integrated interferometers. The observed suppression occurs for a much larger fraction of input–output combinations than what is observed in Fourier interferometers of the same size, and could be relevant to certification of boson sampling machines and other experiments relying on bosonic interference, such as quantum simulation and quantum metrology.

Generation of Constructs for DNA-Directed RNA Interference of Venezuelan Equine Encephalitis Virus Genes

DTIC Science & Technology

2006-12-01

Defence Research and Recherche et developpement Development Canada pour la defense Canada DEFENCE I I! / DEFENSE Generation of Constructs for DNA... research into specific antiviral strategies. One such strategy is RNA interference. RNA interference involves the targeted silencing of a gene using...of an effective vaccine or therapeutic for VEE, a highly infectious virus, underscores the need for research in this area. In addition, the potential

Suppression of AC railway power-line interference in ECG signals recorded by public access defibrillators

PubMed Central

Dotsinsky, Ivan

2005-01-01

Background Public access defibrillators (PADs) are now available for more efficient and rapid treatment of out-of-hospital sudden cardiac arrest. PADs are used normally by untrained people on the streets and in sports centers, airports, and other public areas. Therefore, automated detection of ventricular fibrillation, or its exclusion, is of high importance. A special case exists at railway stations, where electric power-line frequency interference is significant. Many countries, especially in Europe, use 16.7 Hz AC power, which introduces high level frequency-varying interference that may compromise fibrillation detection. Method Moving signal averaging is often used for 50/60 Hz interference suppression if its effect on the ECG spectrum has little importance (no morphological analysis is performed). This approach may be also applied to the railway situation, if the interference frequency is continuously detected so as to synchronize the analog-to-digital conversion (ADC) for introducing variable inter-sample intervals. A better solution consists of rated ADC, software frequency measuring, internal irregular re-sampling according to the interference frequency, and a moving average over a constant sample number, followed by regular back re-sampling. Results The proposed method leads to a total railway interference cancellation, together with suppression of inherent noise, while the peak amplitudes of some sharp complexes are reduced. This reduction has negligible effect on accurate fibrillation detection. Conclusion The method is developed in the MATLAB environment and represents a useful tool for real time railway interference suppression. PMID:16309558

Suppression of AC railway power-line interference in ECG signals recorded by public access defibrillators.

PubMed

Dotsinsky, Ivan

2005-11-26

Public access defibrillators (PADs) are now available for more efficient and rapid treatment of out-of-hospital sudden cardiac arrest. PADs are used normally by untrained people on the streets and in sports centers, airports, and other public areas. Therefore, automated detection of ventricular fibrillation, or its exclusion, is of high importance. A special case exists at railway stations, where electric power-line frequency interference is significant. Many countries, especially in Europe, use 16.7 Hz AC power, which introduces high level frequency-varying interference that may compromise fibrillation detection. Moving signal averaging is often used for 50/60 Hz interference suppression if its effect on the ECG spectrum has little importance (no morphological analysis is performed). This approach may be also applied to the railway situation, if the interference frequency is continuously detected so as to synchronize the analog-to-digital conversion (ADC) for introducing variable inter-sample intervals. A better solution consists of rated ADC, software frequency measuring, internal irregular re-sampling according to the interference frequency, and a moving average over a constant sample number, followed by regular back re-sampling. The proposed method leads to a total railway interference cancellation, together with suppression of inherent noise, while the peak amplitudes of some sharp complexes are reduced. This reduction has negligible effect on accurate fibrillation detection. The method is developed in the MATLAB environment and represents a useful tool for real time railway interference suppression.

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

USDA-ARS?s Scientific Manuscript database

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

The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

PubMed Central

Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

2010-01-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

PubMed

Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

2011-02-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeted Silencing of MART-1 Gene Expression by RNA Interference Enhances the Migration Ability of Uveal Melanoma Cells

PubMed Central

Zhang, Yidan; Jia, Renbing; Wang, Jing; Xu, Xiaofang; Yao, Yuting; Ge, Shengfan; Fan, Xianqun

2013-01-01

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy. PMID:23877836

Delivery of gene silencing agents for breast cancer therapy

PubMed Central

2013-01-01

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

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

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

Li, Jie; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen; Yang, Xi-fei

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we exploremore » the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.« less

RNA interference therapy: a new solution for intracranial atherosclerosis?

PubMed Central

Tang, Tao; Wong, Ka-Sing

2014-01-01

Intracranial atherosclerotic stenosis (ICAS) of a major intracranial artery, especially middle cerebral artery (MCA), is reported to be one leading cause of ischemic stroke throughout the world. Compared with other stroke subtypes, ICAS is associated with a higher risk of recurrent stroke despite aggressive medical therapy. Increased understanding of the pathophysiology of ICAS has highlighted several possible targets for therapeutic interventions. Both luminal stenosis and plaque components of ICAS have been found to be associated with ischemic stroke based a post-mortem study. Recent application of high-resolution magnetic resonance imaging (HRMRI) in evaluating ICAS provides new insight into the vascular biology of plaque morphology and component. High signal on T1-weighted fat-suppressed images (HST1) within MCA plaque of HRMRI, highly suggested of fresh or recent intraplaque hemorrhage, has been found to be associated with ipsilateral brain infarction. Thus, the higher prevalence of intraplaque hemorrhage and neovasculature in symptomatic patients with MCA stenosis may provide a potential target for plaque stabilization. We hypothesize that RNA interference (RNAi) therapy delivered by modified nanoparticles may achieve in vivo biomedical imaging and targeted therapy. With the rapid developments in studies about therapeutic and diagnostic nanomaterials, future studies further exploring the molecular biology of atherosclerosis may provide more drug targets for plaque stabilization. PMID:25333054

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

PubMed

Nevers, Alicia; Doyen, Antonia; Malabat, Christophe; Néron, Bertrand; Kergrohen, Thomas; Jacquier, Alain; Badis, Gwenael

2018-05-18

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Gain suppression involving both stimulated Raman and hyper-Raman photons in two-step stimulated emissions

NASA Astrophysics Data System (ADS)

Deng, Lu; Garrett, W. R.; Payne, M. G.; Moore, M. A.

1997-05-01

We show that multiphoton destructive interference leading to gain suppression can be produced even when two different step-wise stimulated emissions, such as stimulated Raman and hyper-Raman emissions, are included in the interference loop.

Regulation of Rat Hepatic L-Pyruvate Kinase Promoter Composition and Activity by Glucose, n-3 Polyunsaturated Fatty Acids, and Peroxisome Proliferator-activated Receptor-α Agonist*S

PubMed Central

Xu, Jinghua; Christian, Barbara; Jump, Donald B.

2009-01-01

Carbohydrate regulatory element-binding protein (ChREBP), MAX-like factor X(MLX), and hepatic nuclear factor-4α (HNF-4α)are key transcription factors involved in the glucose-mediated induction of hepatic L-type pyruvate kinase (L-PK) gene transcription. n-3 polyunsaturated fatty acids (PUFA) and WY14643 (peroxisome proliferator-activated receptor α (PPARα) agonist) interfere with glucose-stimulated L-PK gene transcription in vivo and in rat primary hepatocytes. Feeding rats a diet containing n-3 PUFA or WY14643 suppressed hepatic mRNAL-PK but did not suppress hepatic ChREBP or HNF-4α nuclear abundance. Hepatic MLX nuclear abundance, however, was suppressed by n-3 PUFA but not WY14643. In rat primary hepatocytes, glucose-stimulated accumulation of mRNALPK and L-PK promoter activity correlated with increased ChREBP nuclear abundance. This treatment also increased L-PK promoter occupancy by RNA polymerase II (RNA pol II), acetylated histone H3 (Ac-H3), and acetylated histone H4 (Ac-H4) but did not significantly impact L-PK promoter occupancy by ChREBP or HNF-4α. Inhibition of L-PK promoter activity by n-3 PUFA correlated with suppressed RNA pol II, Ac-H3, and Ac-H4 occupancy on the L-PK promoter. Although n-3 PUFA transiently suppressed ChREBP and MLX nuclear abundance, this treatment did not impact ChREBP-LPK promoter interaction. HNF4α-LPK promoter interaction was transiently suppressed by n-3 PUFA. Inhibition of L-PK promoter activity by WY14643 correlated with a transient decline in ChREBP nuclear abundance and decreased Ac-H4 interaction with the L-PK promoter. WY14643, however, had no impact on MLX nuclear abundance or HNF4α-LPK promoter interaction. Although overexpressed ChREBP or HNF-4α did not relieve n-3 PUFA suppression of L-PK gene expression, overexpressed MLX fully abrogated n-3 PUFA suppression of L-PK promoter activity and mRNAL-PK abundance. Overexpressed ChREBP, but not MLX, relieved the WY14643 inhibition of L-PK. In conclusion, n-3 PUFA and WY14643/PPARα target different transcription factors to control L-PK gene transcription. MLX, the heterodimer partner for ChREBP, has emerged as a novel target for n-3 PUFA regulation. PMID:16644726

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

Public access defibrillation: Suppression of 16.7 Hz interference generated by the power supply of the railway systems

PubMed Central

Christov, Ivaylo I; Iliev, Georgi L

2005-01-01

Background A specific problem using the public access defibrillators (PADs) arises at the railway stations. Some countries as Germany, Austria, Switzerland, Norway and Sweden are using AC railroad net power-supply system with rated 16.7 Hz frequency modulated from 15.69 Hz to 17.36 Hz. The power supply frequency contaminates the electrocardiogram (ECG). It is difficult to be suppressed or eliminated due to the fact that it considerably overlaps the frequency spectra of the ECG. The interference impedes the automated decision of the PADs whether a patient should be (or should not be) shocked. The aim of this study is the suppression of the 16.7 Hz interference generated by the power supply of the railway systems. Methods Software solution using adaptive filtering method was proposed for 16.7 Hz interference suppression. The optimal performance of the filter is achieved, embedding a reference channel in the PADs to record the interference. The method was tested with ECGs from AHA database. Results The method was tested with patients of normal sinus rhythms, symptoms of tachycardia and ventricular fibrillation. Simulated interference with frequency modulation from 15.69 Hz to 17.36 Hz changing at a rate of 2% per second was added to the ECGs, and then processed by the suggested adaptive filtering. The method totally suppresses the noise with no visible distortions of the original signals. Conclusion The proposed adaptive filter for noise suppression generated by the power supply of the railway systems has a simple structure requiring a low level of computational resources, but a good reference signal as well. PMID:15766390

Androgen receptor (AR) suppresses miRNA-145 to promote renal cell carcinoma (RCC) progression independent of VHL status

PubMed Central

Chen, Yuan; Sun, Yin; Rao, Qun; Xu, Hua; Li, Lei; Chang, Chawnshang

2015-01-01

Mutational inactivation of the VHL tumor suppressor plays key roles in the development of renal cell carcinoma (RCC), and mutated VHL-mediated VEGF induction has become the main target for the current RCC therapy. Here we identified a signal pathway of VEGF induction by androgen receptor (AR)/miRNA-145 as a new target to suppress RCC progression. Mechanism dissection revealed that AR might function through binding to the androgen receptor element (ARE) located on the promoter region of miRNA-145 to suppress p53's ability to induce expression of miRNA-145 that normally suppresses expression of HIF2α/VEGF/MMP9/CCND1. Suppressing AR with AR-shRNA or introducing exogenous miRNA-145 mimic can attenuate RCC progression independent of VHL status. MiR-145 mimic in preclinical RCC orthotopic xenograft mouse model revealed its efficacy in suppression of RCC progression. These results together identified signals by AR-suppressed miRNA-145 as a key player in the RCC progression via regulating HIF2α/VEGF/MMP9/CCND1 expression levels. Blockade of the newly identified signal by AR inhibition or miRNA-145 mimics has promising therapeutic benefit to suppress RCC progression. PMID:26304926

Stroop interference and negative priming (NP) suppression in normal aging.

PubMed

Mayas, J; Fuentes, L J; Ballesteros, S

2012-01-01

Age-related differences in the reduction of Stroop interference were explored by comparing the performance of 18 younger (of mean age: 30.0±3.9 years) and 18 older healthy adults (of mean age: 75±7.2 years) in a color-word Stroop task. The aim of this study was to determine whether a decrease in the efficiency of inhibitory mechanisms associated with aging could account for age-related differences in the ability to suppress a pre-potent response. Participants performed a Stroop task to assess Stroop interference and NP suppression concurrently. Results showed a greater Stroop interference in older than in young adults. On the other hand, the NP effect was only reliable in the younger group, the older group not showing NP suppression. These findings suggest that the slowing hypothesis alone cannot explain this pattern of results and that the age-related differences must also involve an inhibitory breakdown during aging. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Apigenin Inhibits Enterovirus-71 Infection by Disrupting Viral RNA Association with trans-Acting Factors

PubMed Central

Zhang, Wei; Qiao, Haishi; Lv, Yuanzi; Wang, Jingjing; Chen, Xiaoqing; Hou, Yayi; Tan, Renxiang; Li, Erguang

2014-01-01

Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5′-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication. PMID:25330384

Acoustic interference suppression of quartz crystal microbalance sensor arrays utilizing phononic crystals

NASA Astrophysics Data System (ADS)

Chen, Yung-Yu; Huang, Li-Chung; Wang, Wei-Shan; Lin, Yu-Ching; Wu, Tsung-Tsong; Sun, Jia-Hong; Esashi, Masayoshi

2013-04-01

Acoustic interference suppression of quartz crystal microbalance (QCM) sensor arrays utilizing phononic crystals is investigated in this paper. A square-lattice phononic crystal structure is designed to have a complete band gap covering the QCM's resonance frequency. The monolithic sensor array consisting of two QCMs separated by phononic crystals is fabricated by micromachining processes. As a result, 12 rows of phononic crystals with band gap boost insertion loss between the two QCMs by 20 dB and also reduce spurious modes. Accordingly, the phononic crystal is verified to be capable of suppressing the acoustic interference between adjacent QCMs in a sensor array.

Elimination of interference component in Wigner-Ville distribution for the signal with 1/f spectral characteristic.

PubMed

Chan, H L; Lin, J L; Huang, H H; Wu, C P

1997-09-01

A new technique for interference-term suppression in Wigner-Ville distribution (WVD) is proposed for the signal with 1/f spectrum shape. The spectral characteristic of the signal is altered by f alpha filtering before time-frequency analysis and compensated after analysis. With the utilization of the proposed technique in smoothed pseudo Wigner-Ville distribution, an excellent suppression of interference component can be achieved.

The roles of the FGF signal in zebrafish embryos analyzed using constitutive activation and dominant-negative suppression of different FGF receptors.

PubMed

Ota, Satoshi; Tonou-Fujimori, Noriko; Yamasu, Kyo

2009-01-01

The roles of the FGF family growth factors and their receptors (FGFRs) in zebrafish embryos were examined using variously modified versions of the four FGFR genes (fgfr1-4). Constitutively active forms of all of the examined FGFRs (ca-FGFRs) caused dorsalization, brain caudalization, and secondary axis formation, indicating that the main FGF signal transduction downstream of the receptor is highly similar among FGFRs. All of the membrane-bound type of dominant-negative FGFRs (mdn-FGFRs) derived from the four fgfr genes, which interfere with endogenous FGFRs, produced posterior truncation, as previously reported in both Xenopus and zebrafish. mdn-FGFR3c had the strongest effects on embryos, progressively disrupting the posterior structure as the dose increased. At the highest dose, only the forebrain was formed. At lower doses, mdn-FGFR3c mainly suppressed the paraxial mesoderm. The co-injection of mRNA for different mdn-FGFRs and FGFs resulted in diverse suppression spectra of the respective FGFRs against FGFs. Only mdn-FGFR3c severely suppressed all of the FGFs examined. We also examined the effects of the secretory type of dominant-negative FGFRs (sdn-FGFRs), which are released from cells and trap FGF ligands. Only sdn-FGFR3c resulted in the characteristic effect of selectively disrupting the isthmic development, as well as the tailbud. The co-injection of the mRNA for sdn-FGFRs and FGFs suggested that sdn-FGFR3c inhibits FGFs of the FGF8 subfamily, which is consistent with its specific effects on development. We discuss the implications of our findings obtained in the present study.

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

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

PubMed

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

2018-06-01

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

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

PubMed

Kopechek, Jonathan A; Carson, Andrew R; McTiernan, Charles F; Chen, Xucai; Klein, Edwin C; Villanueva, Flordeliza S

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Posttranscriptional regulation of albumin gene expression by branched-chain amino acids in rats with acute liver injury.

PubMed

Kuwahata, Masashi; Kuramoto, Yasuko; Tomoe, Yuka; Sugata, Emi; Segawa, Hiroko; Ito, Mikiko; Oka, Tatsuzo; Miyamoto, Ken-Ichi

2004-12-24

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched-chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepared from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the coding region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver of galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein in p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubated with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase in albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfere with this binding and improve the translation efficiency of albumin mRNA in injured liver.

Comparison of different shielding methods in acquisition of physiological signals.

PubMed

Yanbing Jiang; Ning Ji; Hui Wang; Xueyu Liu; Yanjuan Geng; Peng Li; Shixiong Chen; Guanglin Li

2017-07-01

Power line interference in the surrounding environment could usually introduce many difficulties when collecting and analyzing physiological signals. Since power line interference is usually several orders of amplitude larger than the physiological electrical signals, methods of suppressing power line interference should be considered during the signal acquisition. Many studies used a hardware or software band-stop filter to suppress power line interference but it could easily cause attenuations and distortions to the signal of interest. In this study, two kinds of methods that used different signals to drive the shields of the electrodes were proposed to reduce the impacts of power line interference. Three channels of two physiological signals (ECG and EMG) were simultaneously collected when the electrodes were not shielded (No-Shield), shielded by ground signals (GND-Shield) and shielded by buffered signals of the corresponding electrodes (Active-Shield), respectively, on a custom hardware platform based on TI ADS1299. The results showed that power line interference would be significantly suppressed when using shielding approaches, and the Active-Shield method could achieve the best performance with a power line interference reduction up to 36dB. The study suggested that the Active-Shield method at the analog front-end was a great candidate to reduce power line interference in routine acquisitions of physiological signals.

Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

PubMed Central

Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

2016-01-01

In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

The promises and pitfalls of RNA-interference-based therapeutics

PubMed Central

Castanotto, Daniela; Rossi, John J.

2009-01-01

The discovery that gene expression can be controlled by the Watson–Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence — a process known as RNA interference — has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded. PMID:19158789

Knockdown of RRS1 by lentiviral-mediated RNAi promotes apoptosis and suppresses proliferation of human hepatocellular carcinoma cells.

PubMed

Wang, Jitao; Li, Zhi; Zuo, Changzeng; Xie, Qingfan; Li, Hui; Jia, Junhong; Zhen, Zhongguang; Qi, Ruizhao; Li, Zhiwei; Liu, Dengxiang; Sun, Baijun

2017-10-01

In recent years it was found that the synthesis and biological activity of ribosomes are closely associated with tumor cell growth, tumorigenesis, and malignant transformation. However, the role of regulator of ribosome synthesis 1 (RRS1) in hepatocellular carcinoma (HCC) has not yet been reported. In the present study, we aimed to examine the potential role of RRS1 in tumor cell growth by using a lentivirus-mediated RNA interference (RNAi) system in the HCC cell line SMMC-7721 in vitro. Compared with that of the negative control group (Lv-shCon), the mRNA and protein expression levels of RRS1 in SMMC-7721 cells transfected with Lv-shRRS1 were significantly decreased. Further experiments found that silencing of RRS1 gene expression in SMMC-7721 cells significantly suppressed cell proliferation, inhibited colony formation capacity, increased apoptosis and arrested cells in the G1 phase. These results suggest that the RRS1 gene plays a critical role in cell proliferation, colony formation, cell apoptosis and cell cycle distribution in human HCC cells, and that silencing of RRS1 by RNAi is a promising therapeutic approach for the treatment of HCC, and should be further developed.

Versatile RNA Interference Nanoplatform for Systemic Delivery of RNAs

PubMed Central

2015-01-01

Development of nontoxic, tumor-targetable, and potent in vivo RNA delivery systems remains an arduous challenge for clinical application of RNAi therapeutics. Herein, we report a versatile RNAi nanoplatform based on tumor-targeted and pH-responsive nanoformulas (NFs). The NF was engineered by combination of an artificial RNA receptor, Zn(II)-DPA, with a tumor-targetable and drug-loadable hyaluronic acid nanoparticle, which was further modified with a calcium phosphate (CaP) coating by in situ mineralization. The NF can encapsulate small-molecule drugs within its hydrophobic inner core and strongly secure various RNA molecules (siRNAs, miRNAs, and oligonucleotides) by utilizing Zn(II)-DPA and a robust CaP coating. We substantiated the versatility of the RNAi nanoplatform by demonstrating effective delivery of siRNA and miRNA for gene silencing or miRNA replacement into different human types of cancer cells in vitro and into tumor-bearing mice in vivo by intravenous administration. The therapeutic potential of NFs coloaded with an anticancer drug doxorubicin (Dox) and multidrug resistance 1 gene target siRNA (siMDR) was also demonstrated in this study. NFs loaded with Dox and siMDR could successfully sensitize drug-resistant OVCAR8/ADR cells to Dox and suppress OVCAR8/ADR tumor cell proliferation in vitro and tumor growth in vivo. This gene/drug delivery system appears to be a highly effective nonviral method to deliver chemo- and RNAi therapeutics into host cells. PMID:24779637

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

PubMed

Chen, Shu-Chuan; Jeng, King-Song; Lai, Michael M C

2017-10-15

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. Copyright © 2017 American Society for Microbiology.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

PubMed Central

Chen, Shu-Chuan; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. PMID:28768860

Effect of North Bicyclo[3.1.0]hexane 2'-Deoxypseudosugars on RNA Interference: A Novel Class of siRNA Modification | Center for Cancer Research

Cancer.gov

The inside cover picture shows how siRNAs modified with North bicyclo[3.1.0]hexane 2'-deoxy-pseudosugars are able to activate the RNA interference machinery. The paper confirms that the North conformation is critical for RNAi activity.

Bringing RNA Interference (RNAi) into the High School Classroom

ERIC Educational Resources Information Center

Sengupta, Sibani

2013-01-01

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

Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing

PubMed Central

Salton, Maayan; Kasprzak, Wojciech K.; Voss, Ty; Shapiro, Bruce A.; Poulikakos, Poulikos I.; Misteli, Tom

2015-01-01

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signaling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumors. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumor formation and slows growth of vemurafenib-resistant tumors. Our results identify an intronic mutation as a molecular basis for RNA splicing-mediated RAF inhibitor resistance and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma. PMID:25971842

Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing.

PubMed

Salton, Maayan; Kasprzak, Wojciech K; Voss, Ty; Shapiro, Bruce A; Poulikakos, Poulikos I; Misteli, Tom

2015-05-14

Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.

RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema.

PubMed

Kai, Yoshiro; Tomoda, Koichi; Yoneyama, Hiroyuki; Yoshikawa, Masanori; Kimura, Hiroshi

2015-12-09

Chondroitin sulfate proteoglycans are an important mediators in inflammation and leukocyte trafficking. However, their roles in pulmonary emphysema have not been explored. In a murine model of elastase-induced pulmonary emphysema, we found increased carbohydrate sulfotransferase 3 (CHST3), a specific enzyme that synthesizes chondroitin 6-sulfate proteoglycan (C6SPG). To elucidate the role of C6SPG, we investigated the effect of small interfering RNA (siRNA) targeting CHST3 that inhibits C6SPG-synthesis on the pathogenesis of pulmonary emphysema. Mice were intraperitoneally injected with CHST3 siRNA or negative control siRNA on day0 and 7 after intratracheal instillation of elastase. Histology, respiratory function, glycosaminoglycans (GAGs) content, bronchoalveolar lavage (BAL), elastin staining and gene expressions of tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-9 mRNA were evaluated on day7 and/or day21. CHST3 mRNA increased at day 7 and decreased thereafter in lung. CHST3 siRNA successfully inhibited the expression of CHST3 mRNA throughout the study and this was associated with significant reduction of GAGs and C6SPG. Airway destruction and respiratory function were improved by the treatment with CHST3 siRNA. CHST3 siRNA reduced the number of macrophages both in BAL and lung parenchyma and also suppressed the increased expressions of TNF-α and MMP-9 mRNA. Futhermore, CHST3 siRNA improved the reduction of the elastin in the alveolar walls. CHST3 siRNA diminishes accumulation of excessive macrophages and the mediators, leading to accelerate the functional recovery from airway damage by repair of the elastin network associated with pulmonary emphysema.

Lentivirus-mediated silencing of HOTAIR lncRNA restores gefitinib sensitivity by activating Bax/Caspase-3 and suppressing TGF-α/EGFR signaling in lung adenocarcinoma.

PubMed

Liu, Yuanshun; Jiang, Hua; Zhou, Hongbin; Ying, Xiwang; Wang, Zhehua; Yang, Yang; Xu, Wulin; He, Xujun; Li, Yaqing

2018-03-01

Secondary resistance is a major limitation in the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor treatment of lung cancer. Previous studies have shown that expression of the long non-coding RNA HOX transcript antisense RNA (HOTAIR) is upregulated in lung cancer, which is correlated with metastasis and poor prognosis. However, the precise role of HOTAIR and its effects on gefitinib resistance in human lung adenocarcinoma are not known. To address this issue, in the present study we established a gefitinib-resistant (R)PC-9 human lung adenocarcinoma cell line and examined cell viability with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. We found that gefitinib concentrations <10 µM inhibited the viability of PC-9 but not RPC-9 cells in a dose-dependent manner. Lentivirus-mediated HOTAIR RNA interference induced cell apoptosis and S-phase arrest, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and flow cytometry. Consistent with these observations, HOTAIR suppression was associated with tumor shrinkage and restoration of gefitinib sensitivity in RPC-9 xenograft mice. Immunohistochemical analyses and western blot revealed that HOTAIR silencing resulted in the upregulation of B cell lymphoma 2-associated X protein (Bax), Caspase-3 and transforming growth factor α (TGF-α) and downregulation of EGFR and B cell lymphoma 2 (Bcl-2) levels. These results indicate that HOTAIR normally prevents the activation of Bax/Caspase-3 while inducing TGF-α/EGFR signaling. Thus, targeting HOTAIR may be a novel therapeutic strategy for treating gefitinib-resistant lung adenocarcinoma.

Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

PubMed

Wu, Ximei; Iguchi, Takuma; Itoh, Norio; Okamoto, Kousuke; Takagi, Tatsuya; Tanaka, Keiichi; Nakanishi, Tsuyoshi

2008-01-01

Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

RNA Interference Therapies for an HIV-1 Functional Cure.

PubMed

Scarborough, Robert J; Gatignol, Anne

2017-12-27

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

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes.

PubMed

Olina, A V; Kulbachinskiy, A V; Aravin, A A; Esyunina, D M

2018-05-01

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

Upregulation of EMMPRIN (OX47) in Rat Dorsal Root Ganglion Contributes to the Development of Mechanical Allodynia after Nerve Injury.

PubMed

Wang, Qun; Sun, Yanyuan; Ren, Yingna; Gao, Yandong; Tian, Li; Liu, Yang; Pu, Yanan; Gou, Xingchun; Chen, Yanke; Lu, Yan

2015-01-01

Matrix metalloproteinases (MMPs) are widely implicated in inflammation and tissue remodeling associated with various neurodegenerative diseases and play an important role in nociception and allodynia. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) plays a key regulatory role for MMP activities. However, the role of EMMPRIN in the development of neuropathic pain is not clear. Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunofluorescence were performed to determine the changes of messenger RNA and protein of EMMPRIN/OX47 and their cellular localization in the rat dorsal root ganglion (DRG) after nerve injury. Paw withdrawal threshold test was examined to evaluate the pain behavior in spinal nerve ligation (SNL) model. The lentivirus containing OX47 shRNA was injected into the DRG one day before SNL. The expression level of both mRNA and protein of OX47 was markedly upregulated in ipsilateral DRG after SNL. OX47 was mainly expressed in the extracellular matrix of DRG. Administration of shRNA targeted against OX47 in vivo remarkably attenuated mechanical allodynia induced by SNL. In conclusion, peripheral nerve injury induced upregulation of OX47 in the extracellular matrix of DRG. RNA interference against OX47 significantly suppressed the expression of OX47 mRNA and the development of mechanical allodynia. The altered expression of OX47 may contribute to the development of neuropathic pain after nerve injury.

Generation of siRNA Nanosheets for Efficient RNA Interference

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

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

EPA Science Inventory

The use of RNA interference (RNAi) gene silencing technology, particularly RNAi for pesticidal purposes to control macroorganism pests, is a relatively recent innovation. Post-transcriptional silencing of gene function is a very rapid process where double-stranded RNA (dsRNA) dir...

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.

Inhibition of DNA methyltransferase 1 by RNA interference reverses epithelial-mesenchymal transition in highly metastatic 95D lung cancer cells by inhibiting the Wnt signaling pathway.

PubMed

Bu, Xiancong; Zhang, Xiangyan; Xu, Jinhong; Yang, Heping; Zhou, Xiangdong; Wang, Haijing; Gong, Liang

2018-06-01

Epigenetic modifications serve important roles in non-small cell lung cancer (NSCLC) tumorigenesis; however, the role of DNA methyltransferase 1 (DNMT1) in lung cancer progression remains unclear. In the present study, the expression of DNMT1 in the human NSCLC cell lines 95D (high invasive ability) and 95C (low invasive ability) was analyzed by western blotting. The results demonstrated that the expression of DNMT1 in 95D cells was significantly higher, compared with in 95C cells and small airway epithelial cells. To further define the role of DNMT1 in tumor migration and invasion in NSCLC cells, RNA interference was used to silence DNMT1 expression. Depletion of DNMT1 significantly inhibited 95D cell invasion and migration. In addition, treatment with DNMT1 small interfering RNA resulted in compact cell morphology and significantly increased epithelial marker E-cadherin expression whilst also decreasing the expression of certain mesenchymal markers, including vimentin and fibronectin. Suppression of DNMT1 increased cytoplasmic β-catenin levels while downregulating nuclear β-catenin and Snail, an important regulator of EMT. The results from the present study suggest that the inhibition of DNMT1 reverses the epithelial-mesenchymal transition partly via the inhibition of the Wnt/β-catenin signaling pathway, and therefore inhibits cell migration and invasion. These results indicate that targeting DNMT1 may inhibit tumor metastasis and that DNMT1 is a promising target for the novel treatment of lung cancer.

XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

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

Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail: obika@phs.osaka-u.ac.jp

Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediatedmore » cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.« less

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

PubMed Central

McTiernan, Charles F.; Chen, Xucai; Klein, Edwin C.; Villanueva, Flordeliza S.

2016-01-01

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

An entomopathogenic bacterium, Xenorhabdus nematophila, suppresses expression of antimicrobial peptides controlled by Toll and Imd pathways by blocking eicosanoid biosynthesis.

PubMed

Hwang, Jihyun; Park, Youngjin; Kim, Yonggyun; Hwang, Jihyun; Lee, Daeweon

2013-07-01

Immune-associated genes of the beet armyworm, Spodoptera exigua, were predicted from 454 pyrosequencing transcripts of hemocytes collected from fifth instar larvae challenged with bacteria. Out of 22,551 contigs and singletons, 36% of the transcripts had at least one significant hit (E-value cutoff of 1e-20) and used to predict immune-associated genes implicated in pattern recognition, prophenoloxidase activation, intracellular signaling, and antimicrobial peptides (AMPs). Immune signaling and AMP genes were further confirmed in their expression patterns in response to different types of microbial challenge. To discriminate the AMP expression signaling between Toll and Imd pathways, RNA interference was applied to specifically knockdown each signal pathway; the separate silencing treatments resulted in differential suppression of AMP genes. An entomopathogenic bacterium, Xenorhabdus nematophila, suppressed expression of most AMP genes controlled by Toll and Imd pathways, while challenge with heat-killed X. nematophila induced expression of all AMPs in experimental larvae. Benzylideneacetone (BZA), a metabolite of X. nematophila, suppressed the AMP gene inductions when it was co-injected with the heat-killed X. nematophila. However, arachidonic acid, a catalytic product of PLA2 , significantly reversed the inhibitory effect of BZA on the AMP gene expression. This study suggests that X. nematophila suppresses AMP production controlled by Toll and Imd pathways by inhibiting eicosanoid biosynthesis in S. exigua. © 2013 Wiley Periodicals, Inc.

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

PubMed Central

Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J.; Backofen, Rolf; Marchfelder, Anita

2015-01-01

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco.

PubMed

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N; Marshall, David; Hancock, Robert D; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-12-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

A novel Meloidogyne enterolobii effector MeTCTP promotes parasitism by suppressing programmed cell death in host plants.

PubMed

Zhuo, Kan; Chen, Jiansong; Lin, Borong; Wang, Jing; Sun, Fengxia; Hu, Lili; Liao, Jinling

2017-01-01

Meloidogyne enterolobii is one of the most important plant-parasitic nematodes that can overcome the Mi-1 resistance gene and damage many economically important crops. Translationally controlled tumour protein (TCTP) is a multifunctional protein that exists in various eukaryotes and plays an important role in parasitism. In this study, a novel M. enterolobii TCTP effector, named MeTCTP, was identified and functionally characterized. MeTCTP was specifically expressed within the dorsal gland and was up-regulated during M. enterolobii parasitism. Transient expression of MeTCTP in protoplasts from tomato roots showed that MeTCTP was localized in the cytoplasm of the host cells. Transgenic Arabidopsis thaliana plants overexpressing MeTCTP were more susceptible to M. enterolobii infection than wild-type plants in a dose-dependent manner. By contrast, in planta RNA interference (RNAi) targeting MeTCTP suppressed the expression of MeTCTP in infecting nematodes and attenuated their parasitism. Furthermore, MeTCTP could suppress programmed cell death triggered by the pro-apoptotic protein BAX. These results demonstrate that MeTCTP is a novel plant-parasitic nematode effector that promotes parasitism, probably by suppressing programmed cell death in host plants. © 2016 BSPP and John Wiley & Sons Ltd.

Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment.

PubMed

Baroncelli, Silvia; Pirillo, Maria F; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Degli Antoni, Anna; Galluzzo, Clementina M; Stentarelli, Chiara; Amici, Roberta; Floridia, Marco

2015-07-01

There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According to plasma HIV-RNA levels, three groups were defined: full suppression (target not detected), low-level viremia (target detected but <37 copies/ml), and quantifiable HIV-RNA (≥37 copies/ml). Multivariable logistic regression was used to define determinants of full viral suppression and of quantifiable HIV-RNA. Among 107 women evaluated at a median gestational age of 35 weeks, 90 (84.1%) had HIV-RNA <37 copies/ml. Most of them (59/90, 65.6%) had full suppression, with the remaining (31/90, 34.4%) showing low-level viremia (median: 11.9 copies/ml; IQR 7.4-16.3). Among the 17 women with quantifiable viral load, median HIV-RNA was 109 copies/ml (IQR 46-251), with only one case showing resistance (mutation M184V; rate: 9.1%). In multivariable analyses, women with higher baseline HIV-RNA levels and with hepatitis C virus (HCV) coinfection were significantly more likely to have quantifiable HIV-RNA in late pregnancy. Full viral suppression was significantly more likely with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and significantly less likely with higher HIV-RNA in early pregnancy. No cases of HIV transmission occurred. In conclusion, HIV-infected pregnant women showed a high rate of viral suppression and a low resistance rate before delivery. In most cases no target HIV-RNA was detected in plasma, suggesting a low risk of subsequent virological rebound and development of resistance. Women with high levels of HIV-RNA in early pregnancy and those who have concomitant HCV infection should be considered at higher risk of having quantifiable HIV-RNA at the end of pregnancy.

Techniques Analysis of the Interference Suppression Algorithm in Broadband Aeronautical Multi-carrier Communication System

NASA Astrophysics Data System (ADS)

Li, Dong-xia; Ye, Qian-wen

Out-of-band radiation suppression algorithm must be used efficiently for broadband aeronautical communication system in order not to interfere the operation of the existing systems in aviation L-Band. Based on the simple introduction of the broadband aeronautical multi-carrier communication (B-AMC) system model, several sidelobe suppression techniques in orthogonal frequency multiplexing (OFDM) system are presented and analyzed so as to find a suitable algorithm for B-AMC system in this paper. Simulation results show that raise-cosine function windowing can suppress the out-of-band radiation of B-AMC system effectively.

Molecular Mechanisms of RNA-Targeting by Cas13-containing Type VI CRISPR-Cas Systems.

PubMed

O'Connell, Mitchell

2018-06-22

Prokaryotic adaptive immune systems use CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, include a single protein known as Cas13 (formerly C2c2), that when assembled with a crRNA forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains, is required for degradation of target RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications. Copyright © 2018. Published by Elsevier Ltd.

RNA targeting with CRISPR-Cas13.

PubMed

Abudayyeh, Omar O; Gootenberg, Jonathan S; Essletzbichler, Patrick; Han, Shuo; Joung, Julia; Belanto, Joseph J; Verdine, Vanessa; Cox, David B T; Kellner, Max J; Regev, Aviv; Lander, Eric S; Voytas, Daniel F; Ting, Alice Y; Zhang, Feng

2017-10-12

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

PubMed Central

Shen, Min; Zhu, Kangshun; Cheng, Du; Liu, Zhihao; Shan, Hong

2013-01-01

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe. PMID:23922634

Bi-dimensional empirical mode decomposition based fringe-like pattern suppression in polarization interference imaging spectrometer

NASA Astrophysics Data System (ADS)

Ren, Wenyi; Cao, Qizhi; Wu, Dan; Jiang, Jiangang; Yang, Guoan; Xie, Yingge; Wang, Guodong; Zhang, Sheqi

2018-01-01

Many observers using interference imaging spectrometer were plagued by the fringe-like pattern(FP) that occurs for optical wavelengths in red and near-infrared region. It brings us more difficulties in the data processing such as the spectrum calibration, information retrieval, and so on. An adaptive method based on the bi-dimensional empirical mode decomposition was developed to suppress the nonlinear FP in polarization interference imaging spectrometer. The FP and corrected interferogram were separated effectively. Meanwhile, the stripes introduced by CCD mosaic was suppressed. The nonlinear interferogram background removal and the spectrum distortion correction were implemented as well. It provides us an alternative method to adaptively suppress the nonlinear FP without prior experimental data and knowledge. This approach potentially is a powerful tool in the fields of Fourier transform spectroscopy, holographic imaging, optical measurement based on moire fringe, etc.

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

PubMed Central

Marraffini, Luciano A.; Sontheimer, Erik J.

2010-01-01

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

Next-generation libraries for robust RNA interference-based genome-wide screens

PubMed Central

Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

2015-01-01

Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

Mechanistic study of intertypic nucleoprotein complex formation and its inhibitory effect toward influenza A virus.

PubMed

Narkpuk, Jaraspim; Jaru-Ampornpan, Peera; Subali, Theressa; Bertulfo, Fatima Carla T; Wongthida, Phonphimon; Jongkaewwattana, Anan

2015-11-01

Co-infection of influenza A and B viruses (IAV and IBV) results in marked decreases in IAV replication. Multiple mechanisms have been proposed for this phenomenon. Recently, we reported that IBV nucleoprotein (BNP) alone can suppress IAV replication and proposed an inhibition model in which BNP binds IAV nucleoprotein (ANP) and disrupts IAV polymerase complexes. Here, using mutagenesis and co-immunoprecipitation, we determined the protein motifs mediating the intertypic ANP-BNP complex and showed that it specifically interferes with ANP׳s interaction with the PB2 subunit of the IAV polymerase but not with the other subunit PB1. We further demonstrated that BNP only suppresses growth of IAVs but not other RNA viruses. However, different IAV strains display varied sensitivity toward the BNP׳s inhibitory effect. Together, our data provide mechanistic insights into intertypic nucleoprotein complex formation and highlight the role of BNP as a potential broad-spectrum anti-IAV agent. Copyright © 2015 Elsevier Inc. All rights reserved.

RNA-Interference Pathways Display High Rates of Adaptive Protein Evolution in Multiple Invertebrates

PubMed Central

Palmer, William H.; Hadfield, Jarrod D.; Obbard, Darren J.

2018-01-01

Conflict between organisms can lead to a reciprocal adaptation that manifests as an increased evolutionary rate in genes mediating the conflict. This adaptive signature has been observed in RNA-interference (RNAi) pathway genes involved in the suppression of viruses and transposable elements in Drosophila melanogaster, suggesting that a subset of Drosophila RNAi genes may be locked in an arms race with these parasites. However, it is not known whether rapid evolution of RNAi genes is a general phenomenon across invertebrates, or which RNAi genes generally evolve adaptively. Here we use population genomic data from eight invertebrate species to infer rates of adaptive sequence evolution, and to test for past and ongoing selective sweeps in RNAi genes. We assess rates of adaptive protein evolution across species using a formal meta-analytic framework to combine data across species and by implementing a multispecies generalized linear mixed model of mutation counts. Across species, we find that RNAi genes display a greater rate of adaptive protein substitution than other genes, and that this is primarily mediated by positive selection acting on the genes most likely to defend against viruses and transposable elements. In contrast, evidence for recent selective sweeps is broadly spread across functional classes of RNAi genes and differs substantially among species. Finally, we identify genes that exhibit elevated adaptive evolution across the analyzed insect species, perhaps due to concurrent parasite-mediated arms races. PMID:29437826

Suppression of 9-cis-epoxycarotenoid dioxygenase, which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato.

PubMed

Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

2012-01-01

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

PubMed Central

Ilegems, Erwin; Pick, Horst M.; Vogel, Horst

2002-01-01

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types. PMID:12466560

RNA interference-mediated intrinsic antiviral immunity in invertebrates.

PubMed

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

2013-01-01

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

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Oral Delivery of Double-Stranded RNA in Larvae of the Yellow Fever Mosquito, Aedes aegypti: Implications for Pest Mosquito Control

PubMed Central

Singh, Aditi D.; Wong, Sylvia; Ryan, Calen P.; Whyard, Steven

2013-01-01

RNA interference has already proven itself to be a highly versatile molecular biology tool for understanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsRNA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only induced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited. PMID:24224468

An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

PubMed

Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

2015-02-13

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression.

PubMed

de Ronde, Dryas; Pasquier, Adrien; Ying, Su; Butterbach, Patrick; Lohuis, Dick; Kormelink, Richard

2014-02-01

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance-inducing wild-type strains (NSs(RI) ), amino acid reversions in NSs from resistance-breaking strains (NSs(RB)), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw-mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N-terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs(RI) and NSs(RB). Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs(RI) showed that Avr functionality could not simply be transferred between species. Although deletion of the C-terminal domain rendered NSs completely dysfunctional, only a few single-amino-acid mutations in the C-terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference. © 2013 BSPP AND JOHN WILEY & SONS LTD.

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

Mimicry technology: suppressing small RNA activity in plants.

PubMed

Rubio-Somoza, Ignacio; Manavella, Pablo Andrés

2011-01-01

Small RNA suppression constitutes one of the major difficulties for a full molecular characterization of their specific roles in plants. Taking advantage of the latest insights into the new post-biogenesis layer of regulation in microRNA (miRNA) activity, it is possible to overcome the above-mentioned limitation (Nat Genet 39:1033-1037, 2007). We engineered the IPS1 non-coding RNA to bear a complementary sequence to a given miRNA family, resulting in specific sequestration of RISC complexes. MIMIC technology allows for the constitutive release of all of the potential targets of a miRNA family as well as tissue-specific and inducible suppression of its activity.

RNAi-Mediated Knockdown of vATPase Subunits Affects Survival and Reproduction of Bed Bugs (Hemiptera: Cimicidae).

PubMed

Basnet, Sanjay; Kamble, Shripat T

2018-05-04

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has resurged as one of the most troublesome household pests affecting people across the globe. Bed bug infestations have increased in recent years primarily due to the evolution of insecticide resistance and the insect's ability to hitchhike with travelers. vATPases are one of the most evolutionarily conserved holoenzymes in eukaryotes, which are mainly involved in proton transport across the plasma membranes and intracellular organelles. RNA interference (RNAi) has been developed as a promising tool for insect control. In this study, we used RNAi as an approach to knock down subunits A and E of the vATPase gene of bed bugs. Delivery of 0.2 µg/insect of dsRNA specific to vATPase-A and vATPase-E into female bed bugs dramatically impaired the laying and viability of eggs over time. Injection of the vATPase-E dsRNA decreased survival of the bed bugs over 30 d. Our results also showed that the knockdown of mRNA is highly effective and persistent up to 30 d post injection. This research demonstrated that silencing of the two vATPase subunits A and E offers a potential strategy to suppress bed bug populations.

Strigolactone-Induced Putative Secreted Protein 1 Is Required for the Establishment of Symbiosis by the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

PubMed

Tsuzuki, Syusaku; Handa, Yoshihiro; Takeda, Naoya; Kawaguchi, Masayoshi

2016-04-01

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.

Viral evasion of intracellular DNA and RNA sensing

PubMed Central

Chan, Ying Kai; Gack, Michaela U.

2016-01-01

The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP–AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals. PMID:27174148

Optical interference fringe reduction in frequency-modulation spectroscopy experiments

NASA Astrophysics Data System (ADS)

Hjelme, Dag Roar; Neegard, Steinar; Vartdal, Erling

1995-08-01

We show both theoretically and experimentally that interference fringe signals can always be suppressed to improve the signal-to-noise ratio, provided that the modulation frequency is of the order of the absorption linewidth or higher. Suppression of optical interference fringes by more than 1 order of magnitude and signal-to-noise ratio enhancement of more than 13 dB is demonstrated by use of a proper choice of laser modulation frequency. A further fringe reduction of 10 dB is possible by adjustment of the local oscillator phase.

TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling.

PubMed

Xiong, Fengxiao; Li, Xuejuan; Yang, Zhiying; Wang, Yu; Gong, Wenyan; Huang, Junying; Chen, Cheng; Liu, Peiqing; Huang, Heqing

2016-12-01

RhoA/ROCK can cause renal inflammation and fibrosis in the context of diabetes by activating nuclear factor-κB (NF-κB). TGR5 is known for its role in maintaining metabolic homeostasis and anti-inflammation, which is closely related to NF-κB inhibition. Given that TGR5 is highly enriched in kidney, we aim to investigate the regulatory role of TGR5 on fibronectin (FN) and transforming growth factor-β1 (TGF-β1) in high glucose (HG)-treated rat glomerular mesangial cells (GMCs). Both the factors are closely related to renal inflammations and mediated by NF-κB. Moreover, our study determines whether such regulation is achieved by the inhibition of RhoA/ROCK and the subsequent NF-κB suppression. Polymerase chain reaction was taken to test the mRNA level of TGR5. Western blot was used to measure the protein expressions of TGR5, FN, TGF-β1, p65, IκBα, phospho-MYPT1 (Thr853), and MYPT1. Glutathione S-transferase-pull down and immunofluorescence were conducted to test the activation of RhoA, the distribution of TGR5, and p65, respectively. Electrophoretic mobility shift assay was adopted to measure the DNA binding activity of NF-κB. In GMCs, TGR5 activation or overexpression significantly suppressed FN and TGF-β1 protein expressions, NF-κB, and RhoA/ROCK activation induced by HG or transfection of constitutively active RhoA. By contrast, TGR5 RNA interference caused enhancement of FN, TGF-β1 protein expressions, increase of RhoA/ROCK activation. However, TGR5 cannot suppress RhoA/ROCK activation when a selective Protein kinase A (PKA) inhibitor was used. This study suggests that in HG-treated GMCs, TGR5 significantly suppresses the NF-κB-mediated upregulation of FN and TGF-β1, which are hallmarks of diabetic nephropathy. These functions are closely related to the suppression of RhoA/ROCK via PKA.

[RNA interference library research progress and its application in cancer research].

PubMed

Zhao, Ning; Cai, Li

2013-02-01

RNA interference is a homologous mRNA special degradation phenomenon which is caused by the double-stranded RNA. RNAi library is a pooled library that is artificially constructed using RNAi technology. As RNAi library has made a major breakthrough in the field of genetic research, it has been widely used in the field of medical research, especially in the field of cancer research. This review discussed the research progress of RNAi library and its applications in cancer research.

Maintenance of memory for melodies: Articulation or attentional refreshing?

PubMed

Nees, Michael A; Corrini, Ellen; Leong, Peri; Harris, Joanna

2017-12-01

Past research on the effects of articulatory suppression on working memory for nonverbal sounds has been characterized by discrepant findings, which suggests that multiple mechanisms may be involved in the rehearsal of nonverbal sounds. In two experiments we examined the potential roles of two theoretical mechanisms of verbal working memory-articulatory rehearsal and attentional refreshing-in the maintenance of memory for short melodies. In both experiments, participants performed a same-different melody comparison task. During an 8-s retention interval, interference tasks were introduced to suppress articulatory rehearsal, attentional refreshing, or both. In Experiment 1, only the conditions that featured articulatory suppression resulted in worse memory performance than in a control condition, and the suppression of both attentional refreshing and articulatory rehearsal concurrently did not impair memory more than articulatory suppression alone. Experiment 2 reproduced these findings and also confirmed that the locus of interference was articulatory and not auditory (i.e., the interference was not attributable to the sound of participants' own voices during articulatory suppression). Both experiments suggested that articulatory rehearsal played a role in the maintenance of melodies in memory, whereas attentional refreshing did not. We discuss potential theoretical implications regarding the mechanisms used for the rehearsal of nonverbal sounds in working memory.

Role for apyrases in polar auxin transport in Arabidopsis.

PubMed

Liu, Xing; Wu, Jian; Clark, Greg; Lundy, Stacey; Lim, Minhui; Arnold, David; Chan, Jing; Tang, Wenqiang; Muday, Gloria K; Gardner, Gary; Roux, Stanley J

2012-12-01

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [(3)H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.

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

Suppression in high-order above-threshold ionization: destructive interference from quantum orbits

NASA Astrophysics Data System (ADS)

Lai, Xuan Yang; Quan, Wei; Yu, Shao Gang; Huang, Yi Yi; Liu, Xiao Jun

2018-05-01

We experimentally study the above-threshold ionization (ATI) spectra of noble gas argon in an intense laser field and focus on a novel suppression structure in the high-order ATI (HATI) spectra. It is found that, when a well-documented resonancelike enhancement feature appears in the HATI spectra, a significant suppression structure is followed in a higher energy region of the spectra. The observation is well reproduced by a numerical solution of the time-dependent Schrödinger equation. In terms of quantum-orbit theory, the observed suppression structure can be ascribed to the destructive interference from longer quantum orbits. Furthermore, an intrinsic relation between the ionization suppression and the ionization enhancement in the HATI spectra is well established.

Interference resolution in major depression.

PubMed

Joormann, Jutta; Nee, Derek Evan; Berman, Marc G; Jonides, John; Gotlib, Ian H

2010-03-01

In two experiments, we investigated individual differences in the ability to resolve interference in participants diagnosed with major depressive disorder (MDD). Participants were administered the "Ignore/Suppress" task, a short-term memory task composed of two steps. In Step 1 ("ignore"), participants were instructed to memorize a set of stimuli while ignoring simultaneously presented irrelevant material. In Step 2 ("suppress"), participants were instructed to forget a subset of the previously memorized material. The ability to resolve interference was indexed by response latencies on two recognition tasks in which participants decided whether a probe was a member of the target set. In Step 1, we compared response latencies to probes from the to-be-ignored list with response latencies to nonrecently presented items. In Step 2, we compared response latencies to probes from the to-be-suppressed list with response latencies to nonrecently presented items. The results indicate that, compared with control participants, depressed participants exhibited increased interference in the "suppress" but not in the "ignore" step of the task, when the stimuli were negative words. No group differences were obtained when we presented letters instead of emotional words. These findings indicate that depression is associated with difficulty in removing irrelevant negative material from short-term memory.

Exploring Fusarium head blight disease control by RNA interference

USDA-ARS?s Scientific Manuscript database

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

Compositions and Methods for Inhibiting Gene Expressions

NASA Technical Reports Server (NTRS)

Williams, Loren D. (Inventor); Hsiao, Chiaolong (Inventor); Fang, Po-Yu (Inventor); Williams, Justin (Inventor)

2018-01-01

A combined packing and assembly method that efficiently packs ribonucleic acid (RNA) into virus like particles (VLPs) has been developed. The VLPs can spontaneously assemble and load RNA in vivo, efficiently packaging specifically designed RNAs at high densities and with high purity. In some embodiments the RNA is capable of interference activity, or is a precursor of a RNA capable of causing interference activity. Compositions and methods for the efficient expression, production and purification of VLP-RNAs are provided. VLP-RNAs can be used for the storage of RNA for long periods, and provide the ability to deliver RNA in stable form that is readily taken up by cells.

A Novel Complex-Coefficient In-Band Interference Suppression Algorithm for Cognitive Ultra-Wide Band Wireless Sensors Networks.

PubMed

Xiong, Hailiang; Zhang, Wensheng; Xu, Hongji; Du, Zhengfeng; Tang, Huaibin; Li, Jing

2017-05-25

With the rapid development of wireless communication systems and electronic techniques, the limited frequency spectrum resources are shared with various wireless devices, leading to a crowded and challenging coexistence circumstance. Cognitive radio (CR) and ultra-wide band (UWB), as sophisticated wireless techniques, have been considered as significant solutions to solve the harmonious coexistence issues. UWB wireless sensors can share the spectrum with primary user (PU) systems without harmful interference. The in-band interference of UWB systems should be considered because such interference can severely affect the transmissions of UWB wireless systems. In order to solve the in-band interference issues for UWB wireless sensor networks (WSN), a novel in-band narrow band interferences (NBIs) elimination scheme is proposed in this paper. The proposed narrow band interferences suppression scheme is based on a novel complex-coefficient adaptive notch filter unit with a single constrained zero-pole pair. Moreover, in order to reduce the computation complexity of the proposed scheme, an adaptive complex-coefficient iterative method based on two-order Taylor series is designed. To cope with multiple narrow band interferences, a linear cascaded high order adaptive filter and a cyclic cascaded high order matrix adaptive filter (CCHOMAF) interference suppression algorithm based on the basic adaptive notch filter unit are also presented. The theoretical analysis and numerical simulation results indicate that the proposed CCHOMAF algorithm can achieve better performance in terms of average bit error rate for UWB WSNs. The proposed in-band NBIs elimination scheme can significantly improve the reception performance of low-cost and low-power UWB wireless systems.

A Novel Complex-Coefficient In-Band Interference Suppression Algorithm for Cognitive Ultra-Wide Band Wireless Sensors Networks

PubMed Central

Xiong, Hailiang; Zhang, Wensheng; Xu, Hongji; Du, Zhengfeng; Tang, Huaibin; Li, Jing

2017-01-01

With the rapid development of wireless communication systems and electronic techniques, the limited frequency spectrum resources are shared with various wireless devices, leading to a crowded and challenging coexistence circumstance. Cognitive radio (CR) and ultra-wide band (UWB), as sophisticated wireless techniques, have been considered as significant solutions to solve the harmonious coexistence issues. UWB wireless sensors can share the spectrum with primary user (PU) systems without harmful interference. The in-band interference of UWB systems should be considered because such interference can severely affect the transmissions of UWB wireless systems. In order to solve the in-band interference issues for UWB wireless sensor networks (WSN), a novel in-band narrow band interferences (NBIs) elimination scheme is proposed in this paper. The proposed narrow band interferences suppression scheme is based on a novel complex-coefficient adaptive notch filter unit with a single constrained zero-pole pair. Moreover, in order to reduce the computation complexity of the proposed scheme, an adaptive complex-coefficient iterative method based on two-order Taylor series is designed. To cope with multiple narrow band interferences, a linear cascaded high order adaptive filter and a cyclic cascaded high order matrix adaptive filter (CCHOMAF) interference suppression algorithm based on the basic adaptive notch filter unit are also presented. The theoretical analysis and numerical simulation results indicate that the proposed CCHOMAF algorithm can achieve better performance in terms of average bit error rate for UWB WSNs. The proposed in-band NBIs elimination scheme can significantly improve the reception performance of low-cost and low-power UWB wireless systems. PMID:28587085

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

DOE PAGES

Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia; ...

2016-10-13

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascademore » effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.« less

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

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

Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascademore » effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.« less

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

PubMed Central

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

2004-01-01

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

Pathogenic effects of Rift Valley fever virus NSs gene are alleviated in cultured cells by expressed antiviral short hairpin RNAs.

PubMed

Scott, Tristan; Paweska, Janusz T; Arbuthnot, Patrick; Weinberg, Marc S

2012-01-01

Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, may cause severe hepatitis, encephalitis and haemorrhagic fever in humans. There are currently no available licensed vaccines or therapies to treat the viral infection in humans. RNA interference (RNAi)-based viral gene silencing offers a promising approach to inhibiting replication of this highly pathogenic virus. The small (S) segment of the RVFV tripartite genome carries the genetic determinates for pathogenicity during infection. This segment encodes the non-structural S (NSs) and essential nucleocapsid (N) genes. To advance RNAi-based inhibition of RVFV replication, we designed several Pol III short hairpin RNA (shRNA) expression cassettes against the NSs and N genes, including a multimerized plasmid vector that included four shRNA expression cassettes. Effective target silencing was demonstrated using full- and partial-length target reporter assays, and confirmed by western blot analysis of exogenous N and NSs expression. Small RNA northern blots showed detectable RNAi guide strand formation from single and multimerized shRNA constructs. Using a cell culture model of RVFV replication, shRNAs targeting the N gene decreased intracellular nucleocapsid protein concentration and viral replication. The shRNAs directed against the NSs gene reduced NSs protein concentrations and alleviated NSs-mediated cytotoxicity, which may be caused by host transcription suppression. These data are the first demonstration that RNAi activators have a potential therapeutic benefit for countering RVFV infection.

RNA therapeutics: Beyond RNA interference and antisense oligonucleotides

PubMed Central

Kole, Ryszard; Krainer, Adrian R.; Altman, Sidney

2016-01-01

Here we discuss three RNA therapeutic technologies exploiting various oligonucleotides that bind RNA by base-pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by enzyme-dependent degradation of targeted mRNA. Steric blocking oligonucleotides block access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or also downregulate gene expression. Moreover, they can be extensively chemically modified, resulting in more drug-like properties. The ability of RNA blocking oligonucleotides to restore gene function makes them suited for treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to realizing its clinical potential. PMID:22262036

Modeling RNA interference in mammalian cells

PubMed Central

2011-01-01

Background RNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers. Results We tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects. Conclusions Our model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology. PMID:21272352

Phosphorylation of AKT induced by phosphorylated Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells in vitro.

PubMed

Li, Rujun; Li, Junyang; Sang, Dongping; Lan, Qing

2015-01-01

The aim of this study is to determine whether phosphorylation of AKT could be effected by t-AUCB-induced p-Hsp27 and whether p-AKT inhibition sensitizes glioblastoma cells to t-AUCB, and to evaluate the effects of simultaneous inhibition of p-Hsp27 and p-AKT on t-AUCB treated glioblastoma cells. Cell growth was detected using CCK-8 assay; Caspase-3 activity assay kits and flow cytometry were used in apoptosis analysis; Western blot analysis was used to detect p-Hsp27 and p-AKT levels; RNA interference using the siRNA oligos of Hsp27 was performed to knockdown gene expression of Hsp27. All data were analyzed by the Student-Newman-Keul's test. We demonstrated that t-AUCB treatment induces AKT phosphorylation by activating Hsp27 in U251 and LN443 cell lines. Inhibition of AKT phosphorylation by AKT inhibitor IV sensitizes glioblastoma cells to t-AUCB, strengthens t-AUCB suppressing cell growth and inducing cell apoptosis. We also found inhibiting both p-Hsp27 and p-AKT synergistically strengthen t-AUCB suppressing cell growth. Thus, p-AKT induced by p-Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells. Targeting p-Hsp27 and/or p-AKT may be a potential effective strategy for the treatment of glioblastoma.

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

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

Zhao, Meng; He, Hong-wei; Sun, Huan-xing

2009-09-18

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, wemore » evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.« less

RNA interference: from biology to drugs and therapeutics.

PubMed

Appasani, Krishnarao

2004-07-01

RNA interference (RNAi) is a newly discovered and popular technology platform among researchers not only in the fields of RNA biology and molecular cell biology. It has created excitement in clinical sciences such as oncology, neurology, endocrinology, infectious diseases and drug discovery. There is an urgent need to educate and connect academic and industry researchers for the purpose of knowledge transfer. Thus, GeneExpression Systems of Waltham organized its Second International Conference in Waltham City (May 2-4, 2004, MA, USA) on the theme of 'RNA interference: From Biology to Drugs & Therapeutics.' About 200 participants and 32 speakers attended this two and half-day event which was arranged in six scientific and three technology sessions and ended with a panel discussion. This report covers a few representative talks from academia, biotech and the drug industry.

Respiratory viral diseases: access to RNA interference therapy

PubMed Central

Bitko, Vira; Barik, Sailen

2008-01-01

This review summarizes recent experimental achievements in the area of the development of new RNA interference (RNAi) therapeutics for the treatment of viral respiratory diseases. Delivery of siRNA to their intended target tissue remains the biggest problem for most therapeutic applications of these compounds. Appropriate formulations and chemical modifications for improved stability will boost the probability of utilization of RNAi drugs in the clinical applications. PMID:19081824

Chemical modification: the key to clinical application of RNA interference?

PubMed Central

Corey, David R.

2007-01-01

RNA interference provides a potent and specific method for controlling gene expression in human cells. To translate this potential into a broad new family of therapeutics, it is necessary to optimize the efficacy of the RNA-based drugs. As discussed in this Review, it might be possible to achieve this optimization using chemical modifications that improve their in vivo stability, cellular delivery, biodistribution, pharmacokinetics, potency, and specificity. PMID:18060019

Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo.

PubMed

Chen, Weizao; Liu, Mingqiu; Jiao, Ye; Yan, Weiyao; Wei, Xuefeng; Chen, Jiulian; Fei, Liang; Liu, Yang; Zuo, Xiaoping; Yang, Fugui; Lu, Yonggan; Zheng, Zhaoxin

2006-04-01

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.

The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition*S

PubMed Central

Dissanayake, Samudra K.; Wade, Michael; Johnson, Carrie E.; O’Connell, Michael P.; Leotlela, Poloko D.; French, Amanda D.; Shah, Kavita V.; Hewitt, Kyle J.; Rosenthal, Devin T.; Indig, Fred E.; Jiang, Yuan; Nickoloff, Brian J.; Taub, Dennis D.; Trent, Jeffrey M.; Moon, Randall T.; Bittner, Michael; Weeraratna, Ashani T.

2008-01-01

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/β-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner. PMID:17426020

Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance.

PubMed

Ni, Mi; Ma, Wei; Wang, Xiaofang; Gao, Meijing; Dai, Yan; Wei, Xiaoli; Zhang, Lei; Peng, Yonggang; Chen, Shuyuan; Ding, Lingyun; Tian, Yue; Li, Jie; Wang, Haiping; Wang, Xiaolin; Xu, Guowang; Guo, Wangzhen; Yang, Yihua; Wu, Yidong; Heuberger, Shannon; Tabashnik, Bruce E; Zhang, Tianzhen; Zhu, Zhen

2017-09-01

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant 'pyramids' producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross-resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double-stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH-binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt-resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt-resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non-transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Uncoupling Store-Operated Ca2+ Entry and Altered Ca2+ Release from Sarcoplasmic Reticulum through Silencing of Junctophilin Genes

PubMed Central

Hirata, Yutaka; Brotto, Marco; Weisleder, Noah; Chu, Yi; Lin, Peihui; Zhao, Xiaoli; Thornton, Angela; Komazaki, Shinji; Takeshima, Hiroshi; Ma, Jianjie; Pan, Zui

2006-01-01

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging. PMID:16565048

[RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].

PubMed

Wei, Min; Zhang, Yan-Ling; Chen, Lan; Cai, Cui-Xia; Wang, Han-Duo

2016-02-20

To explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms. Three HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting. Transfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (P<0.01). HERC4 silencing by siRNA-3 markedly suppressed the proliferation and migration of Hela cells, increased the apoptosis rate (P<0.01) and reduced the expression levels of cyclin D1 and Bcl-2 (P<0.01). Silencing of HERC4 efficiently inhibits the proliferation, migration, and invasion of Hela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

Ultraviolet B inhibition of DNMT1 activity via AhR activation dependent SIRT1 suppression in CD4+ T cells from systemic lupus erythematosus patients.

PubMed

Wu, Zhouwei; Mei, Xingyu; Ying, Zuolin; Sun, Yue; Song, Jun; Shi, Weimin

2017-06-01

Previous studies have reported that ultraviolet B (UVB) inhibits DNA methyltransferase1 (DNMT1) activity in CD4+ T cells from systemic lupus erythematosus (SLE) patients. Silent mating type information regulation 2 homolog 1 (SIRT1) is a type of Class III histone deacetylases (HDACs), and has been reported to play roles in the pathogenesis of different autoimmune diseases and can modulate DNMT1 activity. Moreover, aryl hydrocarbon receptor (AhR) has been reported to link UVB with SLE. However, the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells remain largely unknown. To elucidate the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells. Twenty-two newly diagnosed active SLE patients and 30 healthy controls were enrolled in the study. CD4+ T cells were isolated, cultured and treated. DNMT1 activity assay, quantitative real-time PCR (qRT-PCR), Western blotting, RNA interference using small interfering RNA and Chromatin Immunoprecipitation (ChIP) assay were employed. DNMT1 activity was inhibited in si-SIRT1-transfected CD4+ T cells, and increased by the established SIRT1 activator, SRT1720. Moreover, the mRNA and protein expression of SIRT1 were suppressed by UVB exposure in lupus CD4+ T cells. UVB-inhibited DNMT1 activity was reversed by SRT1720 in si-control-transfected lupus CD4+ T cells, but not in si-SIRT1-transfected lupus CD4 + T cells. Furthermore, AhR activation by VAF347 reduced the mRNA and protein expression of SIRT1. ChIP using an antibody against AhR in normal CD4+ T cells revealed a 16-fold stronger signal at the site about 1.6kb upstream from the translation start site of the SIRT1 promoter. Finally, UVB could activate AhR and inhibit the mRNA and protein expression of SIRT1. AhR knockdown abrogated the inhibition of UVB-mediated SIRT1 mRNA and protein expression and DNMT1 activity in lupus CD4+ T cells. UVB suppressed SIRT1 expression via activating AhR, and subsequently inhibited DNMT1 activity in CD4+ T cells from SLE patients. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Nitric oxide mediates antimicrobial peptide gene expression by activating eicosanoid signaling

PubMed Central

Sadekuzzaman, Md.

2018-01-01

Nitric oxide (NO) mediates both cellular and humoral immune responses in insects. Its mediation of cellular immune responses uses eicosanoids as a downstream signal. However, the cross-talk with two immune mediators was not known in humoral immune responses. This study focuses on cross-talk between two immune mediators in inducing gene expression of anti-microbial peptides (AMPs) of a lepidopteran insect, Spodoptera exigua. Up-regulation of eight AMPs was observed in S. exigua against bacterial challenge. However, the AMP induction was suppressed by injection of an NO synthase inhibitor, L-NAME, while little expressional change was observed on injecting its enantiomer, D-NAME. The functional association between NO biosynthesis and AMP gene expression was further supported by RNA interference (RNAi) against NO synthase (SeNOS), which suppressed AMP gene expression under the immune challenge. The AMP induction was also mimicked by NO alone because injecting an NO analog, SNAP, without bacterial challenge significantly induced the AMP gene expression. Interestingly, an eicosanoid biosynthesis inhibitor, dexamethasone (DEX), suppressed the NO induction of AMP expression. The inhibitory activity of DEX was reversed by the addition of arachidonic acid, a precursor of eicosanoid biosynthesis. AMP expression of S. exigua was also controlled by the Toll/IMD signal pathway. The RNAi of Toll receptors or Relish suppressed AMP gene expression by suppressing NO levels and subsequently reducing PLA2 enzyme activity. These results suggest that eicosanoids are a downstream signal of NO mediation of AMP expression against bacterial challenge. PMID:29466449

Conditional RNAi: towards a silent gene therapy.

PubMed

Lee, Sang-Kyung; Kumar, Priti

2009-07-02

RNA interference (RNAi) has the potential to permit the downregulation of virtually any gene. While transgenic RNAi enables stable propagation of the resulting phenotype to progeny, the dominant nature of RNAi limits its use to applications where the continued suppression of gene expression does not disturb normal cell functioning. This is of particular importance when the target gene product is essential for cell survival, development or differentiation. It is therefore desirable that knockdown be externally regulatable. This review is aimed at providing an overview of the approaches for conditional RNAi in mammalian systems, with a special mention of studies employing these approaches to target therapeutically/biologically relevant molecules, their advantages and disadvantages, and a pointer towards approaches best suited for RNAi-based gene therapy.

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

PubMed

Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C

1999-10-15

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

PubMed Central

Hardt, W D; Warnecke, J M; Erdmann, V A; Hartmann, R K

1995-01-01

We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex. Images PMID:7540978

Short hairpin RNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization.

PubMed

Flanagan, Sheryl A; Cooper, Kristin S; Mannava, Sudha; Nikiforov, Mikhail A; Shewach, Donna S

2012-12-01

To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. shRNA suppression of TS was compared with 5-fluoro-2'-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy. Copyright © 2012 Elsevier Inc. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?

PubMed

Shoji, Tsubasa; Hashimoto, Takashi

2008-08-01

Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability.

PubMed

Murugan, Malavika; Harward, Stephen; Scharff, Constance; Mooney, Richard

2013-12-18

Mutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian ortholog FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. Here we show that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms. Copyright © 2013 Elsevier Inc. All rights reserved.

Diminished FoxP2 levels affect dopaminergic modulation of corticostriatal signaling important to song variability

PubMed Central

Murugan, Malavika; Harward, Stephen; Scharff, Constance; Mooney, Richard

2013-01-01

Summary Mutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian orthologue FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. Here we show that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anaesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms. PMID:24268418

Silence of the transcripts: RNA interference in medicine.

PubMed

Barik, Sailen

2005-10-01

Silencing of gene expression by ribonucleic acid (RNA), known as RNA interference (RNAi), is now recognized as a major means of gene regulation in biology. In this mechanism, small noncoding double-stranded RNA molecules knock down gene expression through a variety of mechanisms that include messenger RNA (mRNA) degradation, inhibition of mRNA translation, or chromatin remodeling. The posttranscriptional mechanism of RNAi has been embraced by researchers as a powerful tool for generating deficient phenotypes without mutating the gene. In parallel, exciting recent results have promised its application in disease therapy. This review aims to summarize the current knowledge in this area and provide a roadmap that may eventually launch RNAi from the research bench to the medicine chest.

Gene silencing efficiency and INF-β induction effects of splicing miRNA 155-based artificial miRNA with pre-miRNA stem-loop structures.

PubMed

Sin, Onsam; Mabiala, Prudence; Liu, Ye; Sun, Ying; Hu, Tao; Liu, Qingzhen; Guo, Deyin

2012-02-01

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.

Effects of visual and verbal interference tasks on olfactory memory: the role of task complexity.

PubMed

Annett, J M; Leslie, J C

1996-08-01

Recent studies have demonstrated that visual and verbal suppression tasks interfere with olfactory memory in a manner which is partially consistent with a dual coding interpretation. However, it has been suggested that total task complexity rather than modality specificity of the suppression tasks might account for the observed pattern of results. This study addressed the issue of whether or not the level of difficulty and complexity of suppression tasks could explain the apparent modality effects noted in earlier experiments. A total of 608 participants were each allocated to one of 19 experimental conditions involving interference tasks which varied suppression type (visual or verbal), nature of complexity (single, double or mixed) and level of difficulty (easy, optimal or difficult) and presented with 13 target odours. Either recognition of the odours or free recall of the odour names was tested on one occasion, either within 15 minutes of presentation or one week later. Both recognition and recall performance showed an overall effect for suppression nature, suppression level and time of testing with no effect for suppression type. The results lend only limited support to Paivio's (1986) dual coding theory, but have a number of characteristics which suggest that an adequate account of olfactory memory may be broadly similar to current theories of face and object recognition. All of these phenomena might be dealt with by an appropriately modified version of dual coding theory.

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.

The role of suppression in figurative language comprehension✩

PubMed Central

Gemsbacher, Morton Ann; Robertson, Rachel R.W.

2014-01-01

In this paper, we describe the crucial role that suppression plays in many aspects of language comprehension. We define suppression as a general, cognitive mechanism, the purpose of which is to attenuate the interference caused by the activation of extraneous, unnecessary, or inappropriate information. We illustrate the crucial role that suppression plays in general comprehension by reviewing numerous experiments. These experiments demonstrate that suppression attenuates interference during lexical access (how word meanings are ‘accessed’), anaphoric reference (how referents for anaphors, like pronouns, are computed), cataphoric reference (how concepts that are marked by devices, such as spoken stress, gain a privileged status), syntactic parsing (how grammatical forms of sentences are decoded), and individual differences in (adult) language comprehension skill. We also review research that suggests that suppression plays a crucial role in the understanding of figurative language, in particular, metaphors, idioms, and proverbs. PMID:25520540

Suppression of 9-cis-Epoxycarotenoid Dioxygenase, Which Encodes a Key Enzyme in Abscisic Acid Biosynthesis, Alters Fruit Texture in Transgenic Tomato1[W][OA

PubMed Central

Sun, Liang; Sun, Yufei; Zhang, Mei; Wang, Ling; Ren, Jie; Cui, Mengmeng; Wang, Yanping; Ji, Kai; Li, Ping; Li, Qian; Chen, Pei; Dai, Shengjie; Duan, Chaorui; Wu, Yan; Leng, Ping

2012-01-01

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp). PMID:22108525

Shikonin inhibits oxidized LDL-induced monocyte adhesion by suppressing NFκB activation via up-regulation of PI3K/Akt/Nrf2-dependent antioxidation in EA.hy926 endothelial cells.

PubMed

Huang, Chin-Shiu; Lin, Ai-Hsuan; Yang, Ting-Chun; Liu, Kai-Li; Chen, Haw-Wen; Lii, Chong-Kuei

2015-02-01

Oxidized low-density lipoprotein (oxLDL) is a key contributor to atherogenesis through multiple mechanisms, including the reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NFκB) signaling pathway. Although shikonin, one of the main active components isolated from the Chinese herb Lithospermum erythrorhizon, has been shown to possess cardioprotective, antioxidative, and anti-inflammatory effects, the mechanisms underlying these actions are not well understood. In this study, we used EA.hy926 endothelial-like cells to examine the anti-atherogenic activity of shikonin. Shikonin (0-1 μM) concentration-dependently induced heme oxygenase-1, glutamate cysteine ligase modifier subunit, catalase, superoxide dismutase 1, glutathione peroxidase 1, and glutathione reductase protein and mRNA expression and glutathione content via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/Nrf2 signaling pathway. In the presence of oxLDL (40 μg/ml), shikonin pretreatment reversed oxLDL-induced ROS production, antioxidant response element reporter activity, NFκB nuclear translocation, and intercellular adhesion molecule (ICAM)-1 and E-selectin expression and suppressed the increase of monocyte adhesion to endothelial cells. Nrf2 knockdown by using RNA interference attenuated the ability of shikonin to inhibit oxLDL-induced NFκB DNA binding activity, adhesion molecule expression, and monocyte adhesion. Taken together, these results suggest that shikonin protects against oxLDL-induced endothelial damage by suppressing ROS/NFκB-mediated ICAM-1 and E-selectin expression via up-regulation of PI3K/Akt/Nrf2-dependent antioxidant enzyme expression. Copyright © 2014 Elsevier Inc. All rights reserved.

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

PubMed

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

2015-01-01

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

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

PubMed Central

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

2015-01-01

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

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

Flanagan, Sheryl A., E-mail: sflan@umich.edu; Cooper, Kristin S.; Mannava, Sudha

Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquidmore » chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support the further exploration of TS suppression as a novel radiosensitizing strategy.« less

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

USDA-ARS?s Scientific Manuscript database

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

Trojan Horse Strategy for Non-invasive Interference of Clock Gene in the Oyster Crassostrea gigas.

PubMed

Payton, Laura; Perrigault, Mickael; Bourdineaud, Jean-Paul; Marcel, Anjara; Massabuau, Jean-Charles; Tran, Damien

2017-08-01

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

PubMed Central

Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

2013-01-01

Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit. PMID:23242999

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

PubMed

Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

2017-08-01

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host-HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398-415). © 2017 by the American Association for the Study of Liver Diseases.

Domain motions of Argonaute, the catalytic engine of RNA interference

PubMed Central

Ming, Dengming; Wall, Michael E; Sanbonmatsu, Kevin Y

2007-01-01

Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference. PMID:18053142

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

PubMed

Ming, Dengming; Wall, Michael E; Sanbonmatsu, Kevin Y

2007-11-30

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

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

PubMed Central

Natsuizaka, Mitsuteru; Naganuma, Seiji; Kagawa, Shingo; Ohashi, Shinya; Ahmadi, Azal; Subramanian, Harry; Chang, Sanders; Nakagawa, Kei J.; Ji, Xinjun; Liebhaber, Stephen A.; Klein-Szanto, Andres J.; Nakagawa, Hiroshi

2012-01-01

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis. PMID:22415309

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences.

PubMed

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccas e 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like ( Sil1 ), scavenger receptor class C ( Src ), and scavenger receptor class B ( Srb3 and Srb4 ) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean.

Functional Analysis of RNA Interference-Related Soybean Pod Borer (Lepidoptera) Genes Based on Transcriptome Sequences

PubMed Central

Meng, Fanli; Yang, Mingyu; Li, Yang; Li, Tianyu; Liu, Xinxin; Wang, Guoyue; Wang, Zhanchun; Jin, Xianhao; Li, Wenbin

2018-01-01

RNA interference (RNAi) is useful for controlling pests of agriculturally important crops. The soybean pod borer (SPB) is the most important soybean pest in Northeastern Asia. In an earlier study, we confirmed that the SPB could be controlled via transgenic plant-mediated RNAi. Here, the SPB transcriptome was sequenced to identify RNAi-related genes, and also to establish an RNAi-of-RNAi assay system for evaluating genes involved in the SPB systemic RNAi response. The core RNAi genes, as well as genes potentially involved in double-stranded RNA (dsRNA) uptake were identified based on SPB transcriptome sequences. A phylogenetic analysis and the characterization of these core components as well as dsRNA uptake related genes revealed that they contain conserved domains essential for the RNAi pathway. The results of the RNAi-of-RNAi assay involving Laccase 2 (a critical cuticle pigmentation gene) as a marker showed that genes encoding the sid-like (Sil1), scavenger receptor class C (Src), and scavenger receptor class B (Srb3 and Srb4) proteins of the endocytic pathway were required for SPB cellular uptake of dsRNA. The SPB response was inferred to contain three functional small RNA pathways (i.e., miRNA, siRNA, and piRNA pathways). Additionally, the SPB systemic RNA response may rely on systemic RNA interference deficient transmembrane channel-mediated and receptor-mediated endocytic pathways. The results presented herein may be useful for developing RNAi-mediated methods to control SPB infestations in soybean. PMID:29773992

Honokiol confers immunogenicity by dictating calreticulin exposure, activating ER stress and inhibiting epithelial-to-mesenchymal transition.

PubMed

Liu, Shing-Hwa; Lee, Wen-Jane; Lai, De-Wei; Wu, Sheng-Mao; Liu, Chia-Yu; Tien, Hsing-Ru; Chiu, Chien-Shan; Peng, Yen-Chun; Jan, Yee-Jee; Chao, Te-Hsin; Pan, Hung-Chuan; Sheu, Meei-Ling

2015-04-01

Peritoneal dissemination is a major clinical obstacle in gastrointestinal cancer therapy, and it accounts for the majority of cancer-related mortality. Calreticulin (CRT) is over-expressed in gastric tumors and has been linked to poor prognosis. In this study, immunohistochemistry studies revealed that the up-regulation of CRT was associated with lymph node and distant metastasis in patients with gastric cancer specimens. CRT was significantly down-regulated in highly metastatic gastric cancer cell lines and metastatic animal by Honokiol-treated. Small RNA interference blocking CRT by siRNA-CRT was translocated to the cells in the early immunogenic response to Honokiol. Honokiol activated endoplasmic reticulum (ER) stress and down-regulated peroxisome proliferator-activated receptor-γ (PPARγ) activity resulting in PPARγ and CRT degradation through calpain-II activity, which could be reversed by siRNA-calpain-II. The Calpain-II/PPARγ/CRT axis and interaction evoked by Honokiol could be blocked by gene silencing or pharmacological agents. Both transforming growth factor (TGF)-β1 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced cell migration, invasion and reciprocal down-regulation of epithelial marker E-cadherin, which could be abrogated by siRNA-CRT. Moreover, Honokiol significantly suppressed MNNG-induced gastrointestinal tumor growth and over-expression of CRT in mice. Knockdown CRT in gastric cancer cells was found to effectively reduce growth ability and metastasis in vivo. The present study provides insight into the specific biological behavior of CRT in epithelial-to-mesenchymal transition (EMT) and metastasis. Taken together, our results suggest that the therapeutic inhibition of CRT by Honokiol suppresses both gastric tumor growth and peritoneal dissemination by dictating early translocation of CRT in immunogenic cell death, activating ER stress, and blocking EMT. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

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

Hu, Duanmin; Su, Cunjin; Jiang, Min

There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs,more » siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.« less

Vesiculated Long Non-Coding RNAs: Offshore Packages Deciphering Trans-Regulation between Cells, Cancer Progression and Resistance to Therapies

PubMed Central

Fatima, Farah; Nawaz, Muhammad

2017-01-01

Extracellular vesicles (EVs) are nanosized vesicles secreted from virtually all cell types and are thought to transport proteins, lipids and nucleic acids including non-coding RNAs (ncRNAs) between cells. Since, ncRNAs are central to transcriptional regulation during developmental processes; eukaryotes might have evolved novel means of post-transcriptional regulation by trans-locating ncRNAs between cells. EV-mediated transportation of regulatory elements provides a novel source of trans-regulation between cells. In the last decade, studies were mainly focused on microRNAs; however, functions of long ncRNA (lncRNA) have been much less studied. Here, we review the regulatory roles of EV-linked ncRNAs, placing a particular focus on lncRNAs, how they can foster dictated patterns of trans-regulation in recipient cells. This refers to envisaging novel mechanisms of epigenetic regulation, cellular reprogramming and genomic instability elicited in recipient cells, ultimately permitting the generation of cancer initiating cell phenotypes, senescence and resistance to chemotherapies. Conversely, such trans-regulation may introduce RNA interference in recipient cancer cells causing the suppression of oncogenes and anti-apoptotic proteins; thus favoring tumor inhibition. Collectively, understanding these mechanisms could be of great value to EV-based RNA therapeutics achieved through gene manipulation within cancer cells, whereas the ncRNA content of EVs from cancer patients could serve as non-invasive source of diagnostic biomarkers and prognostic indicators in response to therapies. PMID:29657282

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.

Lipopolysaccharide promotes pulmonary fibrosis in acute respiratory distress syndrome (ARDS) via lincRNA-p21 induced inhibition of Thy-1 expression.

PubMed

Zhou, Wen-Qin; Wang, Peng; Shao, Qiu-Ping; Wang, Jian

2016-08-01

Acute respiratory distress syndrome (ARDS) is a common clinical disorder characterized by pulmonary edema leading to acute lung damage and arterial hypoxemia. Pulmonary fibrosis is a progressive, fibrotic lung disorder, whose pathogenesis in ARDS remains speculative. LincRNA-p21 was a novel regulator of cell proliferation, apoptosis and DNA damage response. This study aims to investigate the effects and mechanism of lincRNA-p21 on pulmonary fibrosis in ARDS. Purified 10 mg/kg LPS was dropped into airways of C57BL/6 mice. Expression levels of lincRNA-p21 and Thy-1 were measured by real-time PCR or western blotting. Proliferation of lung fibroblasts was analyzed by BrdU incorporation assay. Lung and BAL collagen contents were estimated using colorimetric Sircol assay. LincRNA-p21 expression was time-dependently increased and Thy-1 expression was time-dependently reduced in a mouse model of ARDS and in LPS-treated lung fibroblasts. Meanwhile, lung fibroblast proliferation was also time-dependently elevated in LPS-treated lung fibroblasts. In addition, lung fibroblast proliferation could be promoted by lincRNA-p21 overexpression and LPS treatment, however, the elevated lung fibroblast proliferation was further abrogated by Thy-1 overexpression or lincRNA-p21 interference. And Thy-1 interference could elevate cell viability of lung fibroblasts and rescue the reduction of lung fibroblast proliferation induced by lincRNA-p21 interference. Moreover, lincRNA-p21 overexpression dramatically inhibited acetylation of H3 and H4 at the Thy-1 promoter and Thy-1 expression levels in HLF1 cells. Finally, lincRNA-p21 interference rescued LPS-induced increase of lung and BAL collagen contents. LincRNA-p21 could lead to pulmonary fibrosis in ARDS by inhibition of the expression of Thy-1.

Intergenic Transcriptional Interference Is Blocked by RNA Polymerase III Transcription Factor TFIIIB in Saccharomyces cerevisiae

PubMed Central

Korde, Asawari; Rosselot, Jessica M.; Donze, David

2014-01-01

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such “extratranscriptional” activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467–ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins. PMID:24336746

Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo.

PubMed

Lin, Daoshu; Cheng, Qiang; Jiang, Qian; Huang, Yuanyu; Yang, Zheng; Han, Shangcong; Zhao, Yuning; Guo, Shutao; Liang, Zicai; Dong, Anjie

2013-05-21

A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery.

lncRNA NONRATT021972 siRNA Decreases Diabetic Neuropathic Pain Mediated by the P2X3 Receptor in Dorsal Root Ganglia.

PubMed

Peng, Haiying; Zou, Lifang; Xie, Jinyan; Wu, Hong; Wu, Bing; Zhu, Gaochun; Lv, Qiulan; Zhang, Xi; Liu, Shuangmei; Li, Guilin; Xu, Hong; Gao, Yun; Xu, Changshui; Zhang, Chunping; Wang, Shouyu; Xue, Yun; Liang, Shangdong

2017-01-01

Long noncoding RNAs (lncRNAs) participate in physiological and pathophysiological processes. Type 2 diabetes mellitus (T2DM) accounts for more than 90 % of all cases of diabetes mellitus (DM). Diabetic neuropathic pain (DNP) is a common complication of T2DM. The aim of this study was to investigate the effects of lncRNA NONRATT021972 small interference RNA (siRNA) on DNP mediated by the P2X 3 receptor in dorsal root ganglia (DRG). These experiments showed that the expression levels of NONRATT021972 in DRG were increased in the T2DM rat model (intraperitoneal injection of STZ with 30 mg/kg). The concentration of NONRATT021972 in T2DM patient serum was higher compared to control healthy subjects. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in T2DM rats were lower compared to control rats. MWT and TWL in T2DM rats treated with NONRATT021972 siRNA were higher compared with those in T2DM rats. The expression levels of the P2X 3 protein and messenger RNA (mRNA) of T2DM rat DRG were higher compared to the control, while those in T2DM rats treated with NONRATT021972 siRNA were significantly lower compared to T2DM rats. The level of tumor necrosis factor-α (TNF-α) in the serum of T2DM rats treated with NONRATT021972 siRNA was significantly decreased compared with T2DM rats. NONRATT021972 siRNA inhibited the phosphorylation and activation of ERK1/2 in T2DM DRG. Thus, NONRATT021972 siRNA treatment may suppress the upregulated expression and activation of the P2X 3 receptor and reduce the hyperalgesia potentiated by the pro-inflammatory cytokine TNF-α in T2DM rats.

The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

PubMed

Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

2011-04-01

The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii.

PubMed

Du, Zhi-Qiang; Lan, Jiang-Feng; Weng, Yu-Ding; Zhao, Xiao-Fan; Wang, Jin-Xing

2013-07-01

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish. Copyright © 2013 Elsevier Ltd. All rights reserved.

Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions.

PubMed

Picco, Gabriele; Petti, Consalvo; Centonze, Alessia; Torchiaro, Erica; Crisafulli, Giovanni; Novara, Luca; Acquaviva, Andrea; Bardelli, Alberto; Medico, Enzo

2017-03-01

In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in-depth characterization. Using a pipeline designed to suppress stroma-derived signal, we find that RSPO3 "outlier" expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)-RSPO3(e2) fusion and a novel PTPRK(e13)-RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long-term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Prokaryotic Argonautes - variations on the RNA interference theme.

PubMed

van der Oost, John; Swarts, Daan C; Jore, Matthijs M

2014-04-15

The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes.

Prokaryotic Argonautes - variations on the RNA interference theme

PubMed Central

van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

2014-01-01

The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

A status report on RNAi therapeutics

PubMed Central

2010-01-01

Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice. PMID:20615220

Molecular mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition.

PubMed

Koliopoulos, Marios G; Lethier, Mathilde; van der Veen, Annemarthe G; Haubrich, Kevin; Hennig, Janosch; Kowalinski, Eva; Stevens, Rebecca V; Martin, Stephen R; Reis E Sousa, Caetano; Cusack, Stephen; Rittinger, Katrin

2018-05-08

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.

Septins suppress the release of vaccinia virus from infected cells.

PubMed

Pfanzelter, Julia; Mostowy, Serge; Way, Michael

2018-06-19

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization. © 2018 Pfanzelter et al.

A sex-ratio meiotic drive system in Drosophila simulans. II: an X-linked distorter.

PubMed

Tao, Yun; Araripe, Luciana; Kingan, Sarah B; Ke, Yeyan; Xiao, Hailian; Hartl, Daniel L

2007-11-06

The evolution of heteromorphic sex chromosomes creates a genetic condition favoring the invasion of sex-ratio meiotic drive elements, resulting in the biased transmission of one sex chromosome over the other, in violation of Mendel's first law. The molecular mechanisms of sex-ratio meiotic drive may therefore help us to understand the evolutionary forces shaping the meiotic behavior of the sex chromosomes. Here we characterize a sex-ratio distorter on the X chromosome (Dox) in Drosophila simulans by genetic and molecular means. Intriguingly, Dox has very limited coding capacity. It evolved from another X-linked gene, which also evolved de nova. Through retrotransposition, Dox also gave rise to an autosomal suppressor, not much yang (Nmy). An RNA interference mechanism seems to be involved in the suppression of the Dox distorter by the Nmy suppressor. Double mutant males of the genotype dox; nmy are normal for both sex-ratio and spermatogenesis. We postulate that recurrent bouts of sex-ratio meiotic drive and its subsequent suppression might underlie several common features observed in the heterogametic sex, including meiotic sex chromosome inactivation and achiasmy.

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

USDA-ARS?s Scientific Manuscript database

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

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

USDA-ARS?s Scientific Manuscript database

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

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

ERIC Educational Resources Information Center

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

2010-01-01

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

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

ERIC Educational Resources Information Center

Kuldell, Natalie H.

2006-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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

Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation

PubMed Central

Ruiz, Newton; de Abreu, Leonardo Araujo; Parizi, Luís Fernando; Kim, Tae Kwon; Mulenga, Albert; Braz, Gloria Regina Cardoso; Vaz, Itabajara da Silva; Logullo, Carlos

2015-01-01

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis. PMID:26091260

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.

The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide.

PubMed

Nemoto, Keiichirou; Hara, Masamitsu; Suzuki, Masashi; Seki, Hikaru; Muranaka, Toshiya; Mano, Yoshihiro

2009-01-22

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.

Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor

PubMed Central

Everts, Bart; Hussaarts, Leonie; Driessen, Nicole N.; Meevissen, Moniek H.J.; Schramm, Gabriele; van der Ham, Alwin J.; van der Hoeven, Barbara; Scholzen, Thomas; Burgdorf, Sven; Mohrs, Markus; Pearce, Edward J.; Hokke, Cornelis H.; Haas, Helmut; Smits, Hermelijn H.

2012-01-01

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming. PMID:22966004

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

PubMed Central

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

[Suppression of replication of swine parvoviral antisense RNA against the NS PPV gene in swine thyroid gland cells].

PubMed

Voskresenskaia, E P; Miroshnichenko, O I; Ponamareva, T I; Savich, O M; Tikhonenko, T I

1993-01-01

The possibility of suppression of porcine parvovirus (PPV) reproduction in the culture of thyroid gland cells of a swine that contain the integrated genes for asRNA against the nonstructural proteins of the virus has been studied. 10 cell lines with the asRNA genes have been obtained. The line with the maximal number of integrated gene copies was used to inflict with the parvovirus. The expression of asRNA in this cell line was shown to lead to 95% suppression of PPV replication as compared with the control cell line.

Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections

PubMed Central

Li, Wenfeng; Evans, Jay D.; Huang, Qiang; Rodríguez-García, Cristina; Liu, Jie; Hamilton, Michele; Grozinger, Christina M.; Webster, Thomas C.; Su, Songkun

2016-01-01

ABSTRACT Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. IMPORTANCE Given the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encoding nkd gene can suppress the reproduction of N. ceranae and improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices. PMID:27613683

Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections.

PubMed

Li, Wenfeng; Evans, Jay D; Huang, Qiang; Rodríguez-García, Cristina; Liu, Jie; Hamilton, Michele; Grozinger, Christina M; Webster, Thomas C; Su, Songkun; Chen, Yan Ping

2016-11-15

Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. Given the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate that knocking down the honey bee immune repressor-encoding nkd gene can suppress the reproduction of N. ceranae and improve the overall health of honey bees, which highlights the potential role of host-derived and RNAi-based therapeutics in controlling the infections in honey bees. The information obtained from this study will have positive implications for honey bee disease management practices. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

PubMed

Fox, Melanie J; Gao, Hongyu; Smith-Kinnaman, Whitney R; Liu, Yunlong; Mosley, Amber L

2015-01-01

The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.

[RNA interference: biogenesis molecular mechanisms and its applications in cervical cancer].

PubMed

Peralta-Zaragoza, Oscar; Bermúdez-Morales, Víctor Hugo; Madrid-Marina, Vicente

2010-01-01

RNAi (RNA interference) is a natural process by which eukaryotic cells silence gene expression through small interference RNAs (siRNA) which are complementary to messenger RNA (mRNA). In this process, the siRNA that are 21-25 nucleotides long and are known as microRNA (miRNA), either associate with the RNA-induced silencing complex (RISC), which targets and cleaves the complementary mRNAs by the endonucleolytic pathway, or repress the translation. It is also possible to silence exogenous gene expression during viral infections by using DNA templates to transcribe siRNA with properties that are identical to those of bioactive microRNA. Persistent human papillomavirus (HPV) infection is the main etiological agent during cervical cancer development and the HPV E6 and E7 oncogenes, which induce cellular transformation and immortalization, represent strategic targets to be silenced with siRNA. In several in vitro and in vivo studies, it has been demonstrated that the introduction of siRNA directed against the E6 and E7 oncogenes in human tumoral cervical cells transformed by HPV, leads to the efficient silencing of HPV E6 and E7 oncogene expression, which induces the accumulation of the products of the p53 and pRb tumor suppressor genes and activates the mechanism of programmed cell death by apoptosis; thus, the progression of the tumoral growth process may be prevented. The goal of this review is to analyze the microRNA biogenesis process in the silencing of gene expression and to discuss the different protocols for the use of siRNA as a potential gene therapy strategy for the treatment of cervical cancer.

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

Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis

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

Liu, Weiwei; Otkur, Wuxiyar; Li, Lingzhi

Highlights: ► Silibinin protects A431 cells from UVB irradiation-induced apoptosis. ► Up-regulation of the IGF-1R-JNK/ERK pathways by UVB induces cell apoptosis. ► Silibinin inhibits IGF-1R pathways to repress caspase-8-mediated apoptosis. -- Abstract: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), suchmore » as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.« less

Pregnane Glycosides Interfere With Steroidogenic Enzymes to Down-Regulate Corticosteroid Production in Human Adrenocortical H295R Cells

PubMed Central

KOMARNYTSKY, SLAVKO; ESPOSITO, DEBORA; POULEV, ALEXANDER; RASKIN, ILYA

2013-01-01

A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na+/K+ ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase. PMID:23065845

Fenofibrate attenuates diabetic nephropathy in experimental diabetic rat's model via suppression of augmented TGF-β1/Smad3 signaling pathway.

PubMed

Al-Rasheed, Nouf Mohamed; Al-Rasheed, Nawal Mohamed; Al-Amin, Maha Abdelrahman; Hasan, Iman Huesein; Al-Ajmi, Hanaa Najeeb; Mohammad, Raeesa Ahmed; Attia, Hala Aboulfotooh

2016-10-01

Fibrates, the ligands of peroxisome profileferator-activated receptor-α have been shown to have a renal protective action in diabetic nephropathy (DN). This study aimed to elucidate the effect of fenofibrate on renal transforming growth factor-β1 (TGF-β1) and Smad3 in Streptozotocin (STZ)-induced DN. Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (55 mg/kg). Diabetic rats were given fenofibrate (100 mg/kg, p.o.). After 12 weeks, diabetic nephropathy biomarkers were assessed. The mRNA expression of collage I and III, TGF-β1 and Smad3 and were detected by RT-PCR. Fenofibrate reduced significantly serum creatinine, kidney/body weight ratio, serum albumin excretion Collage I & III, TGF-β1 and Smad3 mRNA expression. Our results give further insights into the mechanisms underlying the protective role of fenofibrate in DN, suggesting that interference with TGF-β1/Smad3 signaling pathway may be a useful therapeutic approach to prevent DN.

Suppression of RND3 activity by AES downregulation promotes cancer cell proliferation and invasion.

PubMed

Xia, Hongwei; Li, Mingxing; Chen, Liang; Leng, Weibing; Yuan, Dandan; Pang, Xiaohui; Chen, Liu; Li, Ronghui; Tang, Qiulin; Bi, Feng

2013-05-01

Amino-terminal enhancer of split (AES) is a member of the Groucho/TLE family. Although it has no DNA-binding site, AES can regulate transcriptional activity by interacting with transcriptional factors. Emerging evidence indicates that AES may play an important role in tumor metastasis, but the molecular mechanism is still poorly understood. In this study, we found that knockdown of AES by RNA interference (RNAi) downregulated RND3 expression at the mRNA and protein levels in MDA-MB-231 and HepG2, two cancer cell lines. Furthermore, luciferase assays showed that overexpression of AES significantly enhanced RND3 promoter activity. Moreover, inhibition of AES both in MDA-MB-231 and HepG2 cells by RNAi significantly promoted cell proliferation, cell cycle progression and invasion, consistent with the effects of RNAi-mediated RND3 knockdown in these cells. For the first time, data are presented showing that alteration of the malignant behavior of cancer cells by AES is related to RND3 regulation, and these findings also provide new insights into the mechanism of AES action in regulating tumor malignancy.

The Effect of Aging on the Dynamics of Reactive and Proactive Cognitive Control of Response Interference

PubMed Central

Xiang, Ling; Zhang, Baoqiang; Wang, Baoxi; Jiang, Jun; Zhang, Fenghua; Hu, Zhujing

2016-01-01

A prime-target interference task was used to investigate the effects of cognitive aging on reactive and proactive control after eliminating frequency confounds and feature repetitions from the cognitive control measures. We used distributional analyses to explore the dynamics of the two control functions by distinguishing the strength of incorrect response capture and the efficiency of suppression control. For reactive control, within-trial conflict control and between-trial conflict adaption were analyzed. The statistical analysis showed that there were no reliable between-trial conflict adaption effects for either young or older adults. For within-trial conflict control, the results revealed that older adults showed larger interference effects on mean RT and mean accuracy. Distributional analyses showed that the decline mainly stemmed from inefficient suppression rather than from stronger incorrect responses. For proactive control, older adults showed comparable proactive conflict resolution to young adults on mean RT and mean accuracy. Distributional analyses showed that older adults were as effective as younger adults in adjusting their responses based on congruency proportion information to minimize automatic response capture and actively suppress the direct response activation. The results suggest that older adults were less proficient at suppressing interference after conflict was detected but can anticipate and prevent inference in response to congruency proportion manipulation. These results challenge earlier views that older adults have selective deficits in proactive control but intact reactive control. PMID:27847482

The Effect of Aging on the Dynamics of Reactive and Proactive Cognitive Control of Response Interference.

PubMed

Xiang, Ling; Zhang, Baoqiang; Wang, Baoxi; Jiang, Jun; Zhang, Fenghua; Hu, Zhujing

2016-01-01

A prime-target interference task was used to investigate the effects of cognitive aging on reactive and proactive control after eliminating frequency confounds and feature repetitions from the cognitive control measures. We used distributional analyses to explore the dynamics of the two control functions by distinguishing the strength of incorrect response capture and the efficiency of suppression control. For reactive control, within-trial conflict control and between-trial conflict adaption were analyzed. The statistical analysis showed that there were no reliable between-trial conflict adaption effects for either young or older adults. For within-trial conflict control, the results revealed that older adults showed larger interference effects on mean RT and mean accuracy. Distributional analyses showed that the decline mainly stemmed from inefficient suppression rather than from stronger incorrect responses. For proactive control, older adults showed comparable proactive conflict resolution to young adults on mean RT and mean accuracy. Distributional analyses showed that older adults were as effective as younger adults in adjusting their responses based on congruency proportion information to minimize automatic response capture and actively suppress the direct response activation. The results suggest that older adults were less proficient at suppressing interference after conflict was detected but can anticipate and prevent inference in response to congruency proportion manipulation. These results challenge earlier views that older adults have selective deficits in proactive control but intact reactive control.

3′UTR AU-rich elements (AREs) and the RNA-binding protein Tristetraprolin (TTP) are not required for the LPS-mediated destabilization of phospholipase-Cβ-2 mRNA in murine macrophages

PubMed Central

Shukla, Smita; Elson, Genie; Blackshear, Perry J.; Lutz, Carol S.; Leibovich, S. Joseph

2017-01-01

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of Phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA. To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators and proto-oncogenes. Adenylate and Uridylate (AU)-rich elements (AREs) in 3′UTRs are specific recognition sites for RNA-binding proteins including Tristetraprolin (TTP), HuR and AUF1, and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3′UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed Luciferase expression from this reporter. Luciferase expression from mutant 3′UTR constructs lacking AREs was similarly down-regulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP. LPS suppressed PLCβ-2 expression to the same extent in wild type and TTP−/− macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in wild type and TTP−/− macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages. PMID:28124257

3'UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages.

PubMed

Shukla, Smita; Elson, Genie; Blackshear, Perry J; Lutz, Carol S; Leibovich, S Joseph

2017-04-01

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP -/- ). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP -/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP -/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.

Lactobacillus gasseri OLL2809 and its RNA suppress proliferation of CD4(+) T cells through a MyD88-dependent signalling pathway.

PubMed

Yoshida, Ayako; Yamada, Kiyoshi; Yamazaki, Yasumasa; Sashihara, Toshihiro; Ikegami, Shuuji; Shimizu, Makoto; Totsuka, Mamoru

2011-08-01

Recent studies have shown that probiotics are beneficial in prevention and improvement of inflammatory diseases. Accumulating evidence indicates that probiotics can modulate immune cell responses, although the specific molecular mechanism by which probiotics work remains elusive. Because T cells express receptors for microbial components, we examined whether the probiotic strain Lactobacillus gasseri OLL2809 (LG2809) and its components regulate murine CD4(+) T-cell activation. LG2809, as well as two other Lactobacillus strains, inhibited proliferation of CD4(+) T cells; LG2809 had the strongest suppressive activity among them. RNA isolated from LG2809 was also shown to have suppressive activity. We observed this suppressive effect in the culture of CD4(+) T cells stimulated with anti-CD3/CD28 treatment, suggesting a direct effect on CD4(+) T cells. In contrast, the suppressive effect was not observed for CD4(+) T cells from myeloid differentiation primary response gene 88 (MyD88) protein-deficient mice, and was abrogated in the presence of an anti-oxidant reagent, N-acetyl-cysteine (NAC). These results demonstrate that the suppressive effect of LG2809 and its RNA occurred through a MyD88-dependent signalling pathway and suggest involvement of a reactive oxygen species-dependent mechanism. LG2809 RNA injected subcutaneously suppressed delayed-type-hypersensitivity response in DO11.10 mice, and the suppression was abrogated by treatment with NAC. Collectively, these results suggest that suppression of T-cell proliferation by RNA may be one of the mechanisms when a probiotic bacterial strain exerts suppressive effects on inflammatory responses. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

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

USDA-ARS?s Scientific Manuscript database

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

An enzyme free electrochemical biosensor for sensitive detection of miRNA with a high discrimination factor by coupling the strand displacement reaction and catalytic hairpin assembly recycling.

PubMed

Yao, Juan; Zhang, Zhang; Deng, Zhenghua; Wang, Youqiang; Guo, Yongcan

2017-10-23

An isothermal, enzyme free, ultra-specific and ultra-sensitive protocol for electrochemical detection of miRNAs is proposed based on the toehold-mediated strand displacement reaction (SDR) and non-enzymatic catalytic hairpin reaction (CHA) recycling. The SDR was first triggered only in the presence of target miRNA and this process also affects other miRNA interferences having similar target sequences, thus guaranteeing a high discrimination factor and could be used in rare content miRNA detection with various amounts of interferences having similar target sequences. The output protector strand then triggered enzyme free CHA amplification and generates plenty of hairpin self-assembly products. This process in turn influences SDR equilibrium to move to the right and generates large amounts of protector output to ensure analysis sensitivity. Compared with traditional CHA, our proposed method greatly improved the signal to noise ratio and shows excellent performance in rare miRNA detection with miRNA analogue interference. Under the optimal experimental conditions and using square wave voltammetry, the established biosensor could detect target miRNA-21 down to 30 fM (S/N = 3) with a dynamic range from 100 fM to 2 nM, and discriminate rare target miRNA-21 from mismatched miRNA with high selectivity. This method holds great promise in miRNA detection from human cancer cell lines and would be a versatile and powerful tool for clinical molecular diagnostics.

Using RNA interference to knock down the adhesion protein TES.

PubMed

Griffith, Elen

2007-01-01

RNA interference (RNAi) is a specific and efficient method to knock down protein levels using small interfering RNAs (siRNAs), which target mRNA degradation. RNAi can be used in mammalian cell culture systems to target any protein of interest, and several studies have used this method to knock down adhesion proteins. We used siRNAs to knock down the levels of TES, a focal adhesion protein, in HeLa cells. We demonstrated knockdown of both TES mRNA and TES protein. Although total knockdown of TES was not achieved, the observed reduction in TES protein was sufficient to result in a cellular phenotype of reduced actin stress fibers.

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

PubMed

Pratt, Ashley J; MacRae, Ian J

2009-07-03

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

Reactive and Proactive Interference Control in Adults with Autism Spectrum Disorder across the Lifespan

ERIC Educational Resources Information Center

Lever, Anne G.; Ridderinkhof, K. Richard; Marsman, Maarten; Geurts, Hilde M.

2017-01-01

As a large heterogeneity is observed across studies on interference control in autism spectrum disorder (ASD), research may benefit from the use of a cognitive framework that models specific processes underlying reactive and proactive control of interference. Reactive control refers to the expression and suppression of responses and proactive…

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

PubMed

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

2011-12-01

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

Tectonic-1 contributes to the growth and migration of prostate cancer cells in vitro

PubMed Central

WANG, ZHIJUN; GAO, YI; LIU, YUSHAN; CHEN, JIE; WANG, JUNKAI; GAN, SISHUN; XU, DANFENG; CUI, XINGANG

2015-01-01

Tectonic-1 (TCTN1) is an upstream gene involved in embryonic development. The aim of the present study was to investigate the effect of the TCTN1 gene on the viability and migration of prostate cancer cells. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of TCTN1 in PC-3 and DU145 prostate cancer cells. Cell viability and proliferation were measured using MTT and colony formation assays, and the distribution of cells in phases of the cell cycle was determined using flow cytometry. Cell migration was detected using a Transwell assay. The results demonstrated that TCTN1 was widely expressed in several human prostate cancer cell lines. Knockdown of the TCTN1 gene by RNA interference markedly suppressed cell viability and colony formation in the PC-3 and DU145 cell lines. Cell cycle progression was also arrested by TCTN1 silencing. In addition, knockdown of the TCTN1 gene led to the inhibition of cell migration in the two cell lines. These findings confirmed the direct association between the TCTN1 gene and prostate cancer growth in vitro. With further understanding and clinical investigation, this indicates the potential for future development of a novel marker for early detection and gene therapy for prostate cancer. PMID:26310786

Tunable erbium-doped fiber laser based on optical fiber Sagnac interference loop with angle shift spliced polarization maintaining fibers

NASA Astrophysics Data System (ADS)

Ding, Zhenming; Wang, Zhaokun; Zhao, Chunliu; Wang, Dongning

2018-05-01

In this paper, we propose and experimentally demonstrate a tunable erbium-doped fiber laser (EDFL) with Sagnac interference loop with 45° angle shift spliced polarization maintaining fibers (PMFs). In the Sagnac loop, two PMFs with similar lengths. The Sagnac loop outputs a relatively complex interference spectrum since two beams transmitted in clockwise and counterclockwise encounter at the 3 dB coupler, interfere, and form two interference combs when the light transmitted in the Sagnac loop. The laser will excite and be stable when two interference lines in these two interference combs overlap together. Then by adjusting the polarization controller, the wide wavelength tuning is realized. Experimental results show that stable single wavelength laser can be realized in the wavelength range of 1585 nm-1604 nm under the pump power 157.1 mW. The side-mode suppression ratio is not less than 53.9 dB. The peak power fluctuation is less than 0.29 dB within 30 min monitor time and the side-mode suppression ratio is great than 57.49 dB when the pump power is to 222.7 mW.

Adaptive antenna arrays for weak interfering signals

NASA Technical Reports Server (NTRS)

Gupta, I. J.

1985-01-01

The interference protection provided by adaptive antenna arrays to an Earth station or satellite receive antenna system is studied. The case where the interference is caused by the transmission from adjacent satellites or Earth stations whose signals inadverently enter the receiving system and interfere with the communication link is considered. Thus, the interfering signals are very weak. To increase the interference suppression, one can either decrease the thermal noise in the feedback loops or increase the gain of the auxiliary antennas in the interfering signal direction. Both methods are examined. It is shown that one may have to reduce the noise correlation to impractically low values and if directive auxiliary antennas are used, the auxiliary antenna size may have to be too large. One can, however, combine the two methods to achieve the specified interference suppression with reasonable requirements of noise decorrelation and auxiliary antenna size. Effects of the errors in the steering vector on the adaptive array performance are studied.

2D deblending using the multi-scale shaping scheme

NASA Astrophysics Data System (ADS)

Li, Qun; Ban, Xingan; Gong, Renbin; Li, Jinnuo; Ge, Qiang; Zu, Shaohuan

2018-01-01

Deblending can be posed as an inversion problem, which is ill-posed and requires constraint to obtain unique and stable solution. In blended record, signal is coherent, whereas interference is incoherent in some domains (e.g., common receiver domain and common offset domain). Due to the different sparsity, coefficients of signal and interference locate in different curvelet scale domains and have different amplitudes. Take into account the two differences, we propose a 2D multi-scale shaping scheme to constrain the sparsity to separate the blended record. In the domain where signal concentrates, the multi-scale scheme passes all the coefficients representing signal, while, in the domain where interference focuses, the multi-scale scheme suppresses the coefficients representing interference. Because the interference is suppressed evidently at each iteration, the constraint of multi-scale shaping operator in all scale domains are weak to guarantee the convergence of algorithm. We evaluate the performance of the multi-scale shaping scheme and the traditional global shaping scheme by using two synthetic and one field data examples.

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

PubMed

Parrish, S; Fire, A

2001-10-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

PubMed Central

Parrish, S; Fire, A

2001-01-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed. PMID:11680844

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA.

PubMed Central

Heide, C; Pfeiffer, T; Nolan, J M; Hartmann, R K

1999-01-01

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA. PMID:9917070

Digital Receiver for Microwave Radiometry

NASA Technical Reports Server (NTRS)

Ellingson, Steven W.; Hampson, Grant A.; Johnson, Joel T.

2005-01-01

A receiver proposed for use in L-band microwave radiometry (for measuring soil moisture and sea salinity) would utilize digital signal processing to suppress interfering signals. Heretofore, radio frequency interference has made it necessary to limit such radiometry to a frequency band about 20 MHz wide, centered at .1,413 MHz. The suppression of interference in the proposed receiver would make it possible to expand the frequency band to a width of 100 MHz, thereby making it possible to obtain greater sensitivity and accuracy in measuring moisture and salinity

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

PubMed Central

Ko, Jae-hyeong; Llopis, Paula Montero; Heinritz, Jennifer; Jacobs-Wagner, Christine; Söll, Dieter

2013-01-01

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNAHis have distinctive identity elements, we constructed E. coli tRNAHis CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNAHis CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNAHis CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNAHis CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair. PMID:24386240

Seminal HIV-1 RNA Detection in Heterosexual African Men Initiating Antiretroviral Therapy.

PubMed

Mujugira, Andrew; Coombs, Robert W; Heffron, Renee; Celum, Connie; Ronald, Allan; Mugo, Nelly; Baeten, Jared M

2016-07-15

Intermittent shedding of human immunodeficiency virus type 1 (HIV) in semen occurs despite effective antiretroviral therapy (ART) and suppressed blood HIV-1 RNA levels. We assessed the frequency, magnitude, and correlates of seminal HIV-1 RNA shedding in HIV-1-infected African men initiating ART. Seminal HIV-1 RNA was detected in 24% (37 of 155), 10% (5 of 49), and 11% (8 of 70) of samples collected 0-3, 4-6, and >6 months after ART initiation. When blood HIV-1 levels were suppressed, seminal HIV-1 RNA was detected in 8% (16 of 195), and 82% (13 of 16) had an HIV-1 RNA load of < 1000 copies/mL. Seminal HIV-1 RNA shedding was infrequent and present at low levels in HIV-1-infected African men with suppressed blood HIV-1 RNA. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Gene Silencing in Adult Aedes aegypti Mosquitoes Through Oral Delivery of Double-Stranded RNA

DTIC Science & Technology

2012-01-01

utilization of dsRNA as a bio-insecticide against mosquitoes has only recently begun to be evaluated. Double-stranded RNA targeting chitin syn- thase...double- stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito

EGFP-EGF1-Conjugated PLGA Nanoparticles for Targeted Delivery of siRNA into Injured Brain Microvascular Endothelial Cells for Efficient RNA Interference

PubMed Central

Chen, Chen; Mei, Heng; Shi, Wei; Deng, Jun; Zhang, Bo; Guo, Tao; Wang, Huafang; Hu, Yu

2013-01-01

Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer. PMID:23593330

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

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

PubMed Central

Ramachandran, Shyam; Krishnamurthy, Sateesh; Jacobi, Ashley M.; Wohlford-Lenane, Christine; Behlke, Mark A.; Davidson, Beverly L.

2013-01-01

Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1–3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl− conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. PMID:23624792

Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

PubMed

Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

2014-05-16

Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

An experimental SMI adaptive antenna array for weak interfering signals

NASA Technical Reports Server (NTRS)

Dilsavor, R. L.; Gupta, I. J.

1989-01-01

A modified sample matrix inversion (SMI) algorithm designed to increase the suppression of weak interference is implemented on an existing experimental array system. The algorithm itself is fully described as are a number of issues concerning its implementation and evaluation, such as sample scaling, snapshot formation, weight normalization, power calculation, and system calibration. Several experiments show that the steady state performance (i.e., many snapshots are used to calculate the array weights) of the experimental system compares favorably with its theoretical performance. It is demonstrated that standard SMI does not yield adequate suppression of weak interference. Modified SMI is then used to experimentally increase this suppression by as much as 13dB.

IETS and quantum interference: Propensity rules in the presence of an interference feature

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

Lykkebo, Jacob; Solomon, Gemma C., E-mail: gsolomon@nano.ku.dk; Gagliardi, Alessio

2014-09-28

Destructive quantum interference in single molecule electronics is an intriguing phenomenon; however, distinguishing quantum interference effects from generically low transmission is not trivial. In this paper, we discuss how quantum interference effects in the transmission lead to either low current or a particular line shape in current-voltage curves, depending on the position of the interference feature. Second, we consider how inelastic electron tunneling spectroscopy can be used to probe the presence of an interference feature by identifying vibrational modes that are selectively suppressed when quantum interference effects dominate. That is, we expand the understanding of propensity rules in inelastic electronmore » tunneling spectroscopy to molecules with destructive quantum interference.« less

Blood safety implications of donors using HIV pre-exposure prophylaxis.

PubMed

Seed, C R; Yang, H; Lee, J F

2017-07-01

HIV pre-exposure prophylaxis (PrEP) is the use of one or more antiretroviral medications (in combination) to prevent HIV infection. The most commonly used PrEP medication (Truvada ® , Gilead Sciences, Inc.) acts by inhibiting HIV-1 reverse transcriptase. If someone who is using PrEP unknowingly becomes HIV infected (termed 'PrEP breakthrough infection'), there may be suppressed viral replication resulting in a virus level undetectable by the most sensitive HIV NAT. Failure to seroconvert and seroreversion (loss of previously detectable HIV antibodies) have also both been observed with 2nd, 3rd and 4th generation screening immunoassays, as well as Western blot assays. If such a person was tested in the course of donating blood, the results may therefore be difficult to interpret. The index of suspicion for possible PrEP 'interference' should be highest in the context of concomitant low-level positive or 'greyzone' reactivity on HIV NAT and serological tests, which is an unusual pattern in acutely HIV-infected blood donors. Another possibility is detectable HIV RNA with negative HIV serology (i.e. a potential 'NAT yield' case) but without subsequent HIV seroconversion (or disappearance of HIV RNA). Excluding antiretroviral therapy or PrEP use by the donor in such circumstances would be important. The current rarity of PrEP breakthrough infection indicates that any potential safety risk is likely very small. However, considering the increasing use of PrEP we feel it is prudent for those interpreting HIV donor screening test results to consider the potential for PrEP interference. © 2017 International Society of Blood Transfusion.

Suppression of Quasiparticle Scattering Signals in Bilayer Graphene Due to Layer Polarization and Destructive Interference

NASA Astrophysics Data System (ADS)

Jolie, Wouter; Lux, Jonathan; Pörtner, Mathias; Dombrowski, Daniela; Herbig, Charlotte; Knispel, Timo; Simon, Sabina; Michely, Thomas; Rosch, Achim; Busse, Carsten

2018-03-01

We study chemically gated bilayer graphene using scanning tunneling microscopy and spectroscopy complemented by tight-binding calculations. Gating is achieved by intercalating Cs between bilayer graphene and Ir(111), thereby shifting the conduction band minima below the chemical potential. Scattering between electronic states (both intraband and interband) is detected via quasiparticle interference. However, not all expected processes are visible in our experiment. We uncover two general effects causing this suppression: first, intercalation leads to an asymmetrical distribution of the states within the two layers, which significantly reduces the scanning tunneling spectroscopy signal of standing waves mainly present in the lower layer; second, forward scattering processes, connecting points on the constant energy contours with parallel velocities, do not produce pronounced standing waves due to destructive interference. We present a theory to describe the interference signal for a general n -band material.

Suppression of Quasiparticle Scattering Signals in Bilayer Graphene Due to Layer Polarization and Destructive Interference.

PubMed

Jolie, Wouter; Lux, Jonathan; Pörtner, Mathias; Dombrowski, Daniela; Herbig, Charlotte; Knispel, Timo; Simon, Sabina; Michely, Thomas; Rosch, Achim; Busse, Carsten

2018-03-09

We study chemically gated bilayer graphene using scanning tunneling microscopy and spectroscopy complemented by tight-binding calculations. Gating is achieved by intercalating Cs between bilayer graphene and Ir(111), thereby shifting the conduction band minima below the chemical potential. Scattering between electronic states (both intraband and interband) is detected via quasiparticle interference. However, not all expected processes are visible in our experiment. We uncover two general effects causing this suppression: first, intercalation leads to an asymmetrical distribution of the states within the two layers, which significantly reduces the scanning tunneling spectroscopy signal of standing waves mainly present in the lower layer; second, forward scattering processes, connecting points on the constant energy contours with parallel velocities, do not produce pronounced standing waves due to destructive interference. We present a theory to describe the interference signal for a general n-band material.

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

PubMed

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

2013-08-16

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

The right posterior paravermis and the control of language interference.

PubMed

Filippi, Roberto; Richardson, Fiona M; Dick, Frederic; Leech, Robert; Green, David W; Thomas, Michael S C; Price, Cathy J

2011-07-20

Auditory and written language in humans' comprehension necessitates attention to the message of interest and suppression of interference from distracting sources. Investigating the brain areas associated with the control of interference is challenging because it is inevitable that activation of the brain regions that control interference co-occurs with activation related to interference per se. To isolate the mechanisms that control verbal interference, we used a combination of structural and functional imaging techniques in Italian and German participants who spoke English as a second language. First, we searched structural MRI images of Italian participants for brain regions in which brain structure correlated with the ability to suppress interference from the unattended dominant language (Italian) while processing heard sentences in their weaker language (English). This revealed an area in the posterior paravermis of the right cerebellum in which gray matter density was higher in individuals who were better at controlling verbal interference. Second, we found functional activation in the same region when our German participants made semantic decisions on written English words in the presence of interference from unrelated words in their dominant language (German). This combination of structural and functional imaging therefore highlights the contribution of the right posterior paravermis to the control of verbal interference. We suggest that the importance of this region for language processing has previously been missed because most fMRI studies limit the field of view to increase sensitivity, with the lower part of the cerebellum being the region most likely to be excluded.

SiLEncing SLE: the power and promise of small noncoding RNAs.

PubMed

Rigby, Robert J; Vinuesa, Carola G

2008-09-01

In this study, we outline the evidence suggesting that defects in the RNA silencing machinery can lead to the prototypic systemic autoimmune disease, systemic lupus erythematosus, and describe the potential for RNA interference to provide novel therapeutic agents. Over the last year, a class of small noncoding RNAs--microRNAs--have been shown to play key roles in immune regulation including T-cell selection in the thymus, B cell affinity maturation and selection in germinal centres, and development of regulatory T cells, suggesting that the microRNA machinery may be crucial in the maintenance of immunological tolerance. Two RNA silencing mechanisms have been shown to be involved in lupus pathogenesis: failed Roquin-mediated repression of inducible costimulatory receptors messenger RNA through miR-101 in roquin(san/san) mice and decreased expression of pro-apoptotic molecule and phosphatase and tensin homologue on chromosome 10 in mice transgenic for the miR-17-92 cluster, leading to lymphoproliferation and other lupus manisfestations. MicroRNA array experiments performed on peripheral blood mononuclear cells have revealed different expression profiles in systemic lupus erythematosus patients. RNA interference has also been used ex vivo to silence dysregulated T-cell molecules in cells from systemic lupus erythematosus patients. Dysregulation of the RNA silencing machinery has been implicated in systemic lupus erythematosus pathogenesis. Although microRNA profiling may prove to be a useful diagnostic and prognostic tool for a notoriously heterogeneous disease, manipulation of RNA interference emerges as a powerful and potentially specific means to correct dysregulated gene expression in systemic lupus erythematosus patients.

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

Targeting Pin1 by inhibitor API-1 regulates microRNA biogenesis and suppresses hepatocellular carcinoma development.

PubMed

Pu, Wenchen; Li, Jiao; Zheng, Yuanyuan; Shen, Xianyan; Fan, Xin; Zhou, Jian-Kang; He, Juan; Deng, Yulan; Liu, Xuesha; Wang, Chun; Yang, Shengyong; Chen, Qiang; Liu, Lunxu; Zhang, Guolin; Wei, Yu-Quan; Peng, Yong

2018-01-30

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, but there are few effective treatments. Aberrant microRNA (miRNA) biogenesis is correlated with HCC development. We previously demonstrated that peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) participates in miRNA biogenesis and is a potential HCC treatment target. However, how Pin1 modulates miRNA biogenesis remains obscure. Here, we present in vivo evidence that Pin1 overexpression is directly linked to the development of HCC. Administration with the Pin1 inhibitor (API-1), a specific small molecule targeting Pin1 peptidyl-prolyl isomerase domain and inhibiting Pin1 cis-trans isomerizing activity, suppresses in vitro cell proliferation and migration of HCC cells. But API-1-induced Pin1 inhibition is insensitive to HCC cells with low Pin1 expression and/or low exportin-5 (XPO5) phosphorylation. Mechanistically, Pin1 recognizes and isomerizes the phosphorylated serine-proline motif of phosphorylated XPO5 and passivates phosphorylated XPO5. Pin1 inhibition by API-1 maintains the active conformation of phosphorylated XPO5 and restores XPO5-driven precursor miRNA nuclear-to-cytoplasm export, activating anticancer miRNA biogenesis and leading to both in vitro HCC suppression and HCC suppression in xenograft mice. Experimental evidence suggests that Pin1 inhibition by API-1 up-regulates miRNA biogenesis by retaining active XPO5 conformation and suppresses HCC development, revealing the mechanism of Pin1-mediated miRNA biogenesis and unequivocally supporting API-1 as a drug candidate for HCC therapy, especially for Pin1-overexpressing, extracellular signal-regulated kinase-activated HCC. (Hepatology 2018). © 2018 by the American Association for the Study of Liver Diseases.

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

PubMed

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

2014-04-01

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

Inhibition of X-linked inhibitor of apoptosis protein enhances anti-tumor potency of pure total flavonoids on the growth of leukemic cells

PubMed Central

Wu, Liqiang; Zhang, Xiuxia; Lin, Xiaojie; Wang, Bo; Huang, Chang; Qin, Yao; Lin, Shengyun

2018-01-01

Flavonoids, a vast group of polyphenols widely distributed in plants, are known to possess a range of biological activities and potential anti-tumor effects. X-linked inhibitor of apoptosis protein (XIAP) promotes the progression of leukemia by preventing tumor cells undergoing apoptosis. The present study investigated the potential effects and underlying mechanisms of pure total flavonoids from Citrus paradisi Macfad (PTFC) on human U937 cells, and explored the effects of short hairpin (sh)RNA-mediated XIAP knockdown on the anti-cancer effects of PTFC. Western blotting was used to determine level of apoptosis-associated effectors following PTFC treatment. A lentiviral vector of RNA interference of XIAP gene was constructed to downregulate XIAP expression. MTT assay and flow cytometry were used to determine the effects of PTFC separately or combined with XIAP-shRNA on inhibition and apoptosis of U937 cells, respectively. Treatment with PTFC effectively inhibited leukemic cell proliferation in a dose- and time-dependent manner. PTFC induced apoptosis of U937 cells in a dose-dependent manner, at a particular concentration range, by decreasing XIAP expression levels and activating caspases-3, −7 and −9. PTFC treatment combined with XIAP-shRNA additionally demonstrated a marked increase in cell apoptosis, compared with PTFC or XIAP-shRNA alone (P<0.05). Therefore, these findings suggest that PTFC inhibits growth and induces apoptosis in U937 cells in vitro. Furthermore, suppression of XIAP expression enhances these effects. PMID:29434799

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema

PubMed Central

Hu, Hui; Lu, Hong; He, Zhanping; Han, Xiangjun; Chen, Jing; Tu, Rong

2012-01-01

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 μL shRNA- aquaporin-4 (control group) or siRNA- aquaporin-4 solution (1:800) (RNA interference group) into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25–6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5–4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson's correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient (r = −0.806, P < 0.01). These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracellular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema. PMID:25657707

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema: Correlation with variation in apparent diffusion coefficient.

PubMed

Hu, Hui; Lu, Hong; He, Zhanping; Han, Xiangjun; Chen, Jing; Tu, Rong

2012-07-25

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 μL shRNA- aquaporin-4 (control group) or siRNA- aquaporin-4 solution (1:800) (RNA interference group) into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25-6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5-4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson's correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient (r = -0.806, P < 0.01). These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracellular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema.

Ewing's Sarcoma: Development of RNA Interference-Based Therapy for Advanced Disease

PubMed Central

Simmons, Olivia; Maples, Phillip B.; Senzer, Neil; Nemunaitis, John

2012-01-01

Ewing's sarcoma tumors are associated with chromosomal translocation between the EWS gene and the ETS transcription factor gene. These unique target sequences provide opportunity for RNA interference(i)-based therapy. A summary of RNAi mechanism and therapeutically designed products including siRNA, shRNA and bi-shRNA are described. Comparison is made between each of these approaches. Systemic RNAi-based therapy, however, requires protected delivery to the Ewing's sarcoma tumor site for activity. Delivery systems which have been most effective in preclinical and clinical testing are reviewed, followed by preclinical assessment of various silencing strategies with demonstration of effectiveness to EWS/FLI-1 target sequences. It is concluded that RNAi-based therapeutics may have testable and achievable activity in management of Ewing's sarcoma. PMID:22523703

Symbiont-mediated RNA interference in insects

PubMed Central

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

2016-01-01

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

Adaptive suppression of power line interference in ultra-low field magnetic resonance imaging in an unshielded environment

NASA Astrophysics Data System (ADS)

Huang, Xiaolei; Dong, Hui; Qiu, Yang; Li, Bo; Tao, Quan; Zhang, Yi; Krause, Hans-Joachim; Offenhäusser, Andreas; Xie, Xiaoming

2018-01-01

Power-line harmonic interference and fixed-frequency noise peaks may cause stripe-artifacts in ultra-low field (ULF) magnetic resonance imaging (MRI) in an unshielded environment and in a conductively shielded room. In this paper we describe an adaptive suppression method to eliminate these artifacts in MRI images. This technique utilizes spatial correlation of the interference from different positions, and is realized by subtracting the outputs of the reference channel(s) from those of the signal channel(s) using wavelet analysis and the least squares method. The adaptive suppression method is first implemented to remove the image artifacts in simulation. We then experimentally demonstrate the feasibility of this technique by adding three orthogonal superconducting quantum interference device (SQUID) magnetometers as reference channels to compensate the output of one 2nd-order gradiometer. The experimental results show great improvement in the imaging quality in both 1D and 2D MRI images at two common imaging frequencies, 1.3 kHz and 4.8 kHz. At both frequencies, the effective compensation bandwidth is as high as 2 kHz. Furthermore, we examine the longitudinal relaxation times of the same sample before and after compensation, and show that the MRI properties of the sample did not change after applying adaptive suppression. This technique can effectively increase the imaging bandwidth and be applied to ULF MRI detected by either SQUIDs or Faraday coil in both an unshielded environment and a conductively shielded room.

Suppression of HLA Expression by Lentivirus-mediated Gene Transfer of siRNA Cassettes and In Vivo Chemoselection to Enhance Hematopoietic Stem Cell Transplantation

PubMed Central

Hacke, Katrin; Falahati, Rustom; Flebbe-Rehwaldt, Linda; Kasahara, Noriyuki; Gaensler, Karin M. L.

2010-01-01

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts. PMID:19048410

Discordant CSF/plasma HIV-1 RNA in individuals on virologically suppressive antiretroviral therapy in Western India.

PubMed

Dravid, Ameet N; Natrajan, Kartik; Kulkarni, Milind M; Saraf, Chinmay K; Mahajan, Uma S; Kore, Sachin D; Rathod, Niranjan M; Mahajan, Umakant S; Wadia, Rustom S

2018-02-01

Aim of this study was to estimate the prevalence of cerebrospinal fluid (CSF)/Plasma HIV-1 RNA discordance in virologically suppressed individuals presenting with incident neurologic symptoms.In this retrospective cohort study conducted between March 1, 2009, and March 1, 2017, HIV-1 infected adults exposed to atleast 12 months of antiretroviral therapy (ART) and having plasma viral load (VL) <1000 copies/mL (virologically suppressed) were included. Among these, individuals presenting with neurologic symptoms during follow-up were assessed for CSF/Plasma HIV-1 RNA discordance by measuring HIV-1 RNA in collected plasma and CSF samples. CSF/plasma HIV-1 RNA discordance was defined as either detectable CSF HIV-1 RNA (VL > 20 copies/mL) with an undetectable plasma RNA (complete viral suppression, VL ≤20 copies/mL) or CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma RNA when plasma VL was between 20 and 1000 copies/mL (low-level viremia, LLV).Out of 1584 virologically suppressed patients, 71 (4.4%) presented with incident neurologic symptoms. Twenty out of 71 (28.2%) patients were diagnosed with CSF/Plasma HIV-1 discordance. Median plasma and CSF VL in patients with discordance was 120 [interquartile range (IQR): <20 to 332.5] and 4250 (IQR: 2550.0- 9615.0) copies/mL, respectively. All 9 individuals in which CSF HIV-1 genotypic resistance testing was done showed mutations that would compromise efficacy of prescribed ART regimen. Prevalence of CSF/plasma HIV-1 RNA discordance was higher among neurologically symptomatic patients with plasma LLV as compared with those with complete viral suppression (70% vs 11.8%, P < .001). The risk of discordance was also greater in patients who received protease inhibitor (PI) containing ART (P < .001) and those on ART regimens with central nervous system (CNS) penetration effectiveness (CPE) value <6 (P = .006).CSF/plasma HIV-1 RNA discordance indicates replication of HIV-1 that has adapted to the CNS or has developed antiretroviral drug resistance. Larger studies should be performed to study incidence of discordance in India. This will help in managing patients presenting with neurologic symptoms on suppressive ART with appropriate neuroeffective therapy.

Homo sapiens Systemic RNA Interference-defective-1 Transmembrane Family Member 1 (SIDT1) Protein Mediates Contact-dependent Small RNA Transfer and MicroRNA-21-driven Chemoresistance*

PubMed Central

Elhassan, Mohamed O.; Christie, Jennifer; Duxbury, Mark S.

2012-01-01

Locally initiated RNA interference (RNAi) has the potential for spatial propagation, inducing posttranscriptional gene silencing in distant cells. In Caenorhabditis elegans, systemic RNAi requires a phylogenetically conserved transmembrane channel, SID-1. Here, we show that a human SID-1 orthologue, SIDT1, facilitates rapid, contact-dependent, bidirectional small RNA transfer between human cells, resulting in target-specific non-cell-autonomous RNAi. Intercellular small RNA transfer can be both homotypic and heterotypic. We show SIDT1-mediated intercellular transfer of microRNA-21 to be a driver of resistance to the nucleoside analog gemcitabine in human adenocarcinoma cells. Documentation of a SIDT1-dependent small RNA transfer mechanism and the associated phenotypic effects on chemoresistance in human cancer cells raises the possibility that conserved systemic RNAi pathways contribute to the acquisition of drug resistance. Mediators of non-cell-autonomous RNAi may be tractable targets for novel therapies aimed at improving the efficacy of current cytotoxic agents. PMID:22174421

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis1[OPEN

PubMed Central

Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E.; Chen, Ming; Zhou, Yongming; Yu, Bin

2015-01-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. PMID:26048882

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.

Polycomb repressive complex 1 modifies transcription of active genes

PubMed Central

Pherson, Michelle; Misulovin, Ziva; Gause, Maria; Mihindukulasuriya, Kathie; Swain, Amanda; Dorsett, Dale

2017-01-01

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences. PMID:28782042

Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.

PubMed

Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E; Chen, Ming; Zhou, Yongming; Yu, Bin; Cahoon, Edgar B

2015-08-01

Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of Plant Biologists. All Rights Reserved.

Dual silencing of EGFR and HER2 enhances the sensitivity of gastric cancer cells to gefitinib.

PubMed

Wang, Liying; Zhang, Hongfeng; Zheng, Jiaxin; Wei, Xiaoli; Du, Jingwen; Lu, Haibo; Sun, Qiuying; Zhou, Weiyu; Zhang, Rui; Han, Yu

2018-04-10

Gefitinib exhibits very limited efficacy in gastric cancer (GC). Indeed, the limited clinical results obtained with gefitinib alone justify investigation of additional therapeutic strategies. Here, we demonstrate the importance of EGFR and HER2 in GC malignancy using RNA interference (RNAi). Additionally, we explored the ability of RNAi targeting EGFR and HER2 to enhance the sensitivity of GC cells to gefitinib. Specific small interfering RNAs (siRNAs) significantly inhibited mRNA and protein expression of target genes. EGFR-specific siRNA, EGFR/HER2 siRNAs, and gefitinib inhibited growth and induced apoptosis in GC cell lines in a dose-dependent manner. In contrast, resistance to HER2-siRNA-induced growth inhibition and apoptosis was linked to compensatory activation of EGFR. Moreover, gefitinib dramatically reduced p-EGFR and p-HER2 levels in the cell lines tested, and sensitivity to gefitinib was enhanced through dual silencing of EGFR and HER2 via suppression of AKT and ERK activation. These findings are in agreement with the profound inhibitory effect of gefitinib on activation of both EGFR and HER2. Overall, EGFR/HER2 knockdown by siRNAs further decreased the growth of GC cells treated with gefitinib alone, confirming that single-agent drug targeting does not achieve a maximal biological effect. The combination of gefitinib with EGFR/HER2 siRNAs should be further investigated as a new strategy for the treatment of GC and other EGFR/HER2-dependent cancers. © 2018 Wiley Periodicals, Inc.

Anagrelide represses GATA-1 and FOG-1 expression without interfering with thrombopoietin receptor signal transduction.

PubMed

Ahluwalia, M; Donovan, H; Singh, N; Butcher, L; Erusalimsky, J D

2010-10-01

 Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive.  Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34(+) -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis.  In UT-7/mpl cells, anagrelide (1μm) did not interfere with MPL-mediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3μm) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes.  Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events. © 2010 International Society on Thrombosis and Haemostasis.

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

PubMed

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

2017-11-01

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

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

PubMed

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

2016-11-15

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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

2016-01-01

ABSTRACT Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5′-3′ exoribonuclease XRN1/Pacman on conserved RNA structures in the 3′ untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo. Two reproducible small-RNA hot spots within the 3′ UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3′ SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved. PMID:27581979

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation.

PubMed

Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V

2018-06-01

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation

PubMed Central

Richter, Günther H. S.; Plehm, Stephanie; Fasan, Annette; Rössler, Sabine; Unland, Rebekka; Bennani-Baiti, Idriss M.; Hotfilder, Marc; Löwel, Diana; von Luettichau, Irene; Mossbrugger, Ilona; Quintanilla-Martinez, Leticia; Kovar, Heinrich; Staege, Martin S.; Müller-Tidow, Carsten; Burdach, Stefan

2009-01-01

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2−/−γC−/− mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor. PMID:19289832

Primary cilium suppression by SREBP1c involves distortion of vesicular trafficking by PLA2G3

PubMed Central

Gijs, Hannah Laura; Willemarck, Nicolas; Vanderhoydonc, Frank; Khan, Niamat Ali; Dehairs, Jonas; Derua, Rita; Waelkens, Etienne; Taketomi, Yoshitaka; Murakami, Makoto; Agostinis, Patrizia; Annaert, Wim; Swinnen, Johannes V.

2015-01-01

Distortion of primary cilium formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor sterol regulatory element–binding protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently up-regulated in cellular models of SREBP1c-induced cilium repression was phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyzes the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium, as revealed by aberrant transferrin and Rab11 localization, and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells. PMID:25904332

Interferences in electrochemical hydride generation of hydrogen selenide

NASA Astrophysics Data System (ADS)

Bolea, E.; Laborda, F.; Belarra, M. A.; Castillo, J. R.

2001-12-01

Interferences from Cu(II), Zn(II), Pt(IV), As(III) and nitrate on electrochemical hydride generation of hydrogen selenide were studied using a tubular flow-through generator, flow injection sample introduction and quartz tube atomic absorption spectrometry. Comparison with conventional chemical generation using tetrahydroborate was also performed. Lead and reticulated vitreous carbon (RVC), both in particulate form, were used as cathode materials. Signal supressions up to 60-75%, depending on the cathode material, were obtained in the presence of up to 200 mg l-1 of nitrate due to the competitive reduction of the anion. Interference from As(III) was similar in electrochemical and chemical generation, being related to the quartz tube atomization process. Zinc did not interfere up to Se/Zn ratios 1:100, whereas copper and platinum showed suppression levels up to 50% for Se/interferent ratios 1:100. Total signal suppression was observed in presence of Se/Cu ratios 1:100 when RVC cathodes were used. No memory effects were observed in any case. Scanning electron microscopy and squared wave voltametry studies supported the interference mechanism based on the decomposition of the hydride on the dispersed particles of the reduced metal.

Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm.

PubMed

Davis-Vogel, Courtney; Ortiz, Angel; Procyk, Lisa; Robeson, Jonathan; Kassa, Adane; Wang, Yiwei; Huang, Emily; Walker, Carl; Sethi, Amit; Nelson, Mark E; Sashital, Dipali G

2018-05-18

Western corn rootworm (Diabrotica virgifera virgifera) is a serious agricultural pest known for its high adaptability to various management strategies, giving rise to a continual need for new control options. Transgenic maize expressing insecticidal RNAs represents a novel mode of action for rootworm management that is dependent on the RNA interference (RNAi) pathways of the insect for efficacy. Preliminary evidence suggests that western corn rootworm could develop broad resistance to all insecticidal RNAs through changes in RNAi pathway genes; however, the likelihood of field-evolved resistance occurring through this mechanism remains unclear. In the current study, eight key genes involved in facilitating interference in the microRNA and small interfering RNA pathways were targeted for knockdown in order to evaluate impact on fitness of western corn rootworm. These genes include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2. Depletion of targeted transcripts in rootworm larvae led to changes in microRNA expression, decreased ability to pupate, reduced adult beetle emergence, and diminished reproductive capacity. The observed effects do not support evolution of resistance through changes in expression of these eight genes due to reduced insect fitness.

Intersystem Interference Reduction for Overlaid HAPS-Terrestrial CDMA System

NASA Astrophysics Data System (ADS)

Huang, Jeng-Ji; Wang, Wei-Ting; Li, Mingfu; Shiung, David; Ferng, Huei-Wen

In this letter, we propose that directional antennas, combined with power management, be incorporated to reduce intersystem interference in a shared band overlaid high altitude platform station (HAPS)-terrestrial code division multiple access (CDMA) system. To eliminate the HAPS to terrestrial interference, the HAPS is accessed only via directional antennas under the proposed scheme. By doing so, the uplink power to the HAPS can accordingly be increased, so that the terrestrial to HAPS interference is also effectively suppressed.

The WRKY45-2 WRKY13 WRKY42 Transcriptional Regulatory Cascade Is Required for Rice Resistance to Fungal Pathogen1[OPEN

PubMed Central

Cheng, Hongtao; Liu, Hongbo; Deng, Yong; Xiao, Jinghua; Li, Xianghua; Wang, Shiping

2015-01-01

Blast caused by fungal Magnaporthe oryzae is a devastating disease of rice (Oryza sativa) worldwide, and this fungus also infects barley (Hordeum vulgare). At least 11 rice WRKY transcription factors have been reported to regulate rice response to M. oryzae either positively or negatively. However, the relationships of these WRKYs in the rice defense signaling pathway against M. oryzae are unknown. Previous studies have revealed that rice WRKY13 (as a transcriptional repressor) and WRKY45-2 enhance resistance to M. oryzae. Here, we show that rice WRKY42, functioning as a transcriptional repressor, suppresses resistance to M. oryzae. WRKY42-RNA interference (RNAi) and WRKY42-overexpressing (oe) plants showed increased resistance and susceptibility to M. oryzae, accompanied by increased or reduced jasmonic acid (JA) content, respectively, compared with wild-type plants. JA pretreatment enhanced the resistance of WRKY42-oe plants to M. oryzae. WRKY13 directly suppressed WRKY42. WRKY45-2, functioning as a transcriptional activator, directly activated WRKY13. In addition, WRKY13 directly suppressed WRKY45-2 by feedback regulation. The WRKY13-RNAi WRKY45-2-oe and WRKY13-oe WRKY42-oe double transgenic lines showed increased susceptibility to M. oryzae compared with WRKY45-2-oe and WRKY13-oe plants, respectively. These results suggest that the three WRKYs form a sequential transcriptional regulatory cascade. WRKY42 may negatively regulate rice response to M. oryzae by suppressing JA signaling-related genes, and WRKY45-2 transcriptionally activates WRKY13, whose encoding protein in turn transcriptionally suppresses WRKY42 to regulate rice resistance to M. oryzae. PMID:25624395

Regulation of Bach2 by the aryl hydrocarbon receptor as a mechanism for suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

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

De Abrew, K. Nadira; Phadnis, Ashwini S.; Genetics Program, Michigan State University, East Lansing, MI 48824

2011-04-15

Exposure to the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation and suppresses antibody production. Previous genomic studies in mouse B cells identified Bach2 as a direct target of the AHR. Bach2 is known to repress expression of Prdm1, a key transcription factor involved in B-cell differentiation, by binding to Maf elements (MAREs) in the regulatory regions of the gene. Chromatin immunoprecipitation followed by quantitative PCR in TCDD-treated lipopolysaccharide (LPS)-activated B cells showed increased binding of the AHR within the first intron in the Bach2 gene. The binding was further confirmed by electrophoretic mobility shift assay (EMSA). TCDDmore » also induced expression of Bach2 in activated as well as resting B cells from 2 to 24 h post-treatment in a time- and concentration-dependent manner. Expression of Prdm1 was decreased by TCDD at 24 h and was consistent with repression by Bach2. Increased DNA binding activity to the intron 5 MARE with increasing TCDD concentrations was observed by EMSA. Supershifts identified the presence of Bach2 in the DNA binding complex associated with the intron 5 MARE of Prdm1. Functional validation of the role of Bach2 in the suppression of B-cell differentiation by TCDD was performed using RNA interference (RNAi). Knockdown of Bach2 showed approximately 40% reversal in the TCDD-induced suppression of IgM secretion when compared to controls. The results suggest that the transcriptional regulation of Bach2 by the AHR is one of the mechanisms involved in the suppression of B-cell differentiation by TCDD.« less

Tegumental Phosphodiesterase SmNPP-5 Is a Virulence Factor for Schistosomes ▿

PubMed Central

Bhardwaj, Rita; Krautz-Peterson, Greice; Da'dara, Akram; Tzipori, Saul; Skelly, Patrick J.

2011-01-01

The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease that constitutes a major health problem globally. In this study we cloned and characterized the schistosome tegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDa protein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of the definitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis, SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization by immuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegument; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays. To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knock down expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-time PCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, the suppression and the ectolocalization of this enzyme in live parasites were evident because of a significantly impaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substrate p-nitrophenyl 5′-dTMP (p-Nph-5′-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivo were tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 gene was demonstrably suppressed at the time of host infection were greatly impaired in their ability to establish infection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes. PMID:21825060

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

PubMed

Smyth, Redmond P; Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe; von Kleist, Max; Marquet, Roland

2018-05-18

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging

PubMed Central

Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe

2018-01-01

Abstract Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production. PMID:29514260

[Innate immune responses against viral infection and its suppression by viral proteins].

PubMed

Oshiumi, Hiroyuki; Matsumoto, Misako; Seya, Tsukasa

2013-01-01

Retinoic acid-inducible gene-I(RIG-I) is a cytoplasmic RNA helicase and a viral RNA sensor. RIG-I recognizes 5' triphosphate double-stranded RNA (dsRNA) and activates the IPS-1 adaptor molecule. The association of IPS-1 with RIG-I causes the formation of the prion-like structure of IPS-1. This structure is essential for activation of the signaling required for the induction of type I interferon (IFN), which possesses strong antiviral activity. Recent studies have revealed the novel factors involved in the RIG-I-dependent pathway. DDX3 and DDX60 RNA helicases associate with RIG-I and promote its binding to viral RNA. Riplet and TRIM25 ubiquitin ligase deliver Lys63-linked polyubiquitin moiety to RIG-I and result in signal activation. Several pathogenic viruses have evolved excellent systems to suppress type I IFN production. For example, NS3-4A of hepatitis C virus (HCV) cleaves IPS-1, which is the adaptor molecule of RIG-I, while the HCV core protein abrogates DDX3 function to suppress RIG-I-dependent IPS-1 activation, and the NS-1 of flu inhibits TRIM25 function to suppress RIG-I activation.

Nanotherapeutics Using an HIV-1 Poly A and Transactivator of the HIV-1 LTR-(TAR-) Specific siRNA

PubMed Central

Mahajan, Supriya D.; Aalinkeel, Ravikumar; Reynolds, Jessica L.; Nair, Bindukumar; Sykes, Donald E.; Law, Wing-Cheung; Ding, Hong; Bergey, Earl J.; Prasad, Paras N.; Schwartz, Stanley A.

2011-01-01

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. We used a well-validated siRNA (si510) which targets the poly A/TAR (transactivator of the HIV-1 LTR) site and suppresses viral replication. Nanotechnology holds much potential for impact in the field of HIV-1 therapeutics, and nanoparticles such as quantum rods (QRs) can be easily functionalized to incorporate siRNA forming stable nanoplexes that can be used for gene silencing. We evaluated the efficacy of the QR-si510 HIV-1 siRNA nanoplex in suppressing viral replication in the HIV-1-infected monocytic cell line THP-1 by measuring p24 antigen levels and gene expression levels of HIV-1 LTR. Our results suggest that the QR-si510 HIV-1 siRNA nanoplex is not only effective in delivering siRNA, but also in suppressing HIV-1 viral replication for a longer time period. HIV-1 nanotherapeutics can thus enhance systemic bioavailability and offer multifunctionality. PMID:21660279

Messenger RNA transcripts

Treesearch

Dan Cullen

2004-01-01

In contrast to DNA, messenger RNA (mRNA) in complex substrata is rarely analyzed, in large part because labile RNA molecules are difficult to purify. Nucleic acid extractions from fungi that colonize soil are particularly difficult and plagued by humic substances that interfere with Taq polymerase (Tebbe and Vahjen 1993 and references therein). Magnetic capture...

siRNA-loaded cationic liposomes for cancer therapy: Development, characterization and efficacy evaluation

NASA Astrophysics Data System (ADS)

Ying, Bo

Cancer is a major health problem in the United States and many other parts of the world. However, cancer treatment is severely limited by the lack of highly effective cytotoxic agents and selective delivery methods which can serve as the "magic bullet" (first raised by Dr. Paul Ehrlich, the goal of targeting a specific location without causing harm to surrounding tissues or to more distant regions in the body). The revolutionary finding that tumors cannot grow beyond a microscopic size without dedicated blood supply provided a highly effective alternative for the treatment of cancer. Currently, anti-angiogenic therapy and the discovery of RNA interference makes it possible to treat some conditions by silencing disorder-causing genes of targeting cells which are otherwise difficult to eradicate with more conventional therapies. However, before siRNA technology could be widely used as a therapeutic approach, the construct must be efficiently and safely delivered to target cells. Strategies used for siRNA delivery should minimize uptake by phagocytes, enzymatic degradation by nucleases and should be taken up preferentially, if not specifically, by the intended cell population. Kinesin spindle proteins (KSP) are the motor proteins which play critical roles during mitosis. Different from tubulins which are also present in post-mitotic cells, such as axons, KSP is exclusively expressed in mitotic cells, which makes them the ideal target for anti-mitotics. In the present study, we intend to develop, characterize and evaluate a liposome-based delivery system which can deliver KSP siRNA selectively to the tumor vasculature (thus inhibiting angiogenesis, destroying tumor vasculature and eventually, eradicating tumor growth). We first developed ten different liposome preparation types with different compositions of lipids. Next, the capacity for loading siRNA and efficiency of targeting the tumor vascular supply was evaluated using relevant cellular and tumor models. Pegylated cationic liposomes (PCLs) were selected as carriers for siRNA. Based on the silencing efficiency of siRNA formulated with different PCLs, DOPC based cationic liposomes, over DOPE based nanosystems, with a modest amount of polyetheleneglycol was selected to deliver KSP siRNA to tumor-bearing mice. Efficacy studies revealed that tumor suppression was observed when KSP siRNA was delivered using PCLs, but not in mice that received naked KSP siRNA or KSP siRNA in commercially available transfecting agents. The results were further supported by MRI (magnetic resonance imaging) analysis. To evaluate the role that vasculature supply plays in the development of the tumor, we also performed tumor response studies using a tumor model consisting of tumor cells which are resistant to KSP siRNA. The results showed that a prolonged suppression of tumor growth was achieved only when a large dose (5mg/kg) KSP siRNA was administered, but not with the administration of a relatively low dose (2mg/kg) of siRNA, suggesting that a combined treatment approach containing both anti-vasculature and anti-cancer agents should be considered to achieve the best treatment outcome. Finally, it was confirmed by qRT-PCR that the tumor growth inhibition was due to the successful knock-down of KSP mRNA.

RNA interference-based nanosystems for inflammatory bowel disease therapy

PubMed Central

Guo, Jian; Jiang, Xiaojing; Gui, Shuangying

2016-01-01

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is a chronic, recrudescent disease that invades the gastrointestinal tract, and it requires surgery or lifelong medicinal therapy. The conventional medicinal therapies for IBD, such as anti-inflammatories, glucocorticoids, and immunosuppressants, are limited because of their systemic adverse effects and toxicity during long-term treatment. RNA interference (RNAi) precisely regulates susceptibility genes to decrease the expression of proinflammatory cytokines related to IBD, which effectively alleviates IBD progression and promotes intestinal mucosa recovery. RNAi molecules generally include short interfering RNA (siRNA) and microRNA (miRNA). However, naked RNA tends to degrade in vivo as a consequence of endogenous ribonucleases and pH variations. Furthermore, RNAi treatment may cause unintended off-target effects and immunostimulation. Therefore, nanovectors of siRNA and miRNA were introduced to circumvent these obstacles. Herein, we introduce non-viral nanosystems of RNAi molecules and discuss these systems in detail. Additionally, the delivery barriers and challenges associated with RNAi molecules will be discussed from the perspectives of developing efficient delivery systems and potential clinical use. PMID:27789943

Using RNA Interference to Reveal Genetic Vulnerabilities in Human Cancer Cells

DTIC Science & Technology

2005-07-01

pl of RNase/DNase free water and performed PCR amplification in 50pl reaction volumes using Invitrogen’s Platinum® Pfx DNA Polymerase . To obtain a...destroyed1’ 2. This pathway, known as RNA interference (RNAi), has been exploited in organisms ranging from plants to fungi to animals for...experimentally alter its targeting capability. Indeed such strategies have previously succeeded in both plants and animals23󈧜. My initial studies

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

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

Xiong Ruyi; Wu Jianxiang; Zhou Yijun

2009-04-25

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

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

Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference

PubMed Central

Murphy, Katherine A.; Tabuloc, Christine A.; Cervantes, Kevin R.; Chiu, Joanna C.

2016-01-01

RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally found growing on the surface of fruit crops, constitutes a major component of the Drosophila microbiome, and is highly attractive to Drosophila. Thus, this naturally attractive yeast biopesticide can deliver dsRNA to an insect pest without the need for genetic crop modification. We demonstrate that this biopesticide decreases larval survivorship, and reduces locomotor activity and reproductive fitness in adults, which are indicative of general health decline. To our knowledge, this is the first study to show that yeast can be used to deliver dsRNA to an insect pest. PMID:26931800

RNAimmuno: A database of the nonspecific immunological effects of RNA interference and microRNA reagents

PubMed Central

Olejniczak, Marta; Galka-Marciniak, Paulina; Polak, Katarzyna; Fligier, Andrzej; Krzyzosiak, Wlodzimierz J.

2012-01-01

The RNAimmuno database was created to provide easy access to information regarding the nonspecific effects generated in cells by RNA interference triggers and microRNA regulators. Various RNAi and microRNA reagents, which differ in length and structure, often cause non-sequence-specific immune responses, in addition to triggering the intended sequence-specific effects. The activation of the cellular sensors of foreign RNA or DNA may lead to the induction of type I interferon and proinflammatory cytokine release. Subsequent changes in the cellular transcriptome and proteome may result in adverse effects, including cell death during therapeutic treatments or the misinterpretation of experimental results in research applications. The manually curated RNAimmuno database gathers the majority of the published data regarding the immunological side effects that are caused in investigated cell lines, tissues, and model organisms by different reagents. The database is accessible at http://rnaimmuno.ibch.poznan.pl and may be helpful in the further application and development of RNAi- and microRNA-based technologies. PMID:22411954

RNAimmuno: a database of the nonspecific immunological effects of RNA interference and microRNA reagents.

PubMed

Olejniczak, Marta; Galka-Marciniak, Paulina; Polak, Katarzyna; Fligier, Andrzej; Krzyzosiak, Wlodzimierz J

2012-05-01

The RNAimmuno database was created to provide easy access to information regarding the nonspecific effects generated in cells by RNA interference triggers and microRNA regulators. Various RNAi and microRNA reagents, which differ in length and structure, often cause non-sequence-specific immune responses, in addition to triggering the intended sequence-specific effects. The activation of the cellular sensors of foreign RNA or DNA may lead to the induction of type I interferon and proinflammatory cytokine release. Subsequent changes in the cellular transcriptome and proteome may result in adverse effects, including cell death during therapeutic treatments or the misinterpretation of experimental results in research applications. The manually curated RNAimmuno database gathers the majority of the published data regarding the immunological side effects that are caused in investigated cell lines, tissues, and model organisms by different reagents. The database is accessible at http://rnaimmuno.ibch.poznan.pl and may be helpful in the further application and development of RNAi- and microRNA-based technologies.

Towards the elements of successful insect RNAi.

PubMed

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

2013-12-01

RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells.

PubMed

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W

2015-01-01

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

PubMed Central

Bryniarski, Krzysztof; Ptak, Wlodzimierz; Martin, Emilia; Nazimek, Katarzyna; Szczepanik, Marian; Sanak, Marek; Askenase, Philip W.

2015-01-01

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases. PMID:25923429

Runx2- and histone deacetylase 3-mediated repression is relieved in differentiating human osteoblast cells to allow high bone sialoprotein expression.

PubMed

Lamour, Virginie; Detry, Cédric; Sanchez, Christelle; Henrotin, Yves; Castronovo, Vincent; Bellahcène, Akeila

2007-12-14

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

PubMed Central

Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

2015-01-01

Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

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

USDA-ARS?s Scientific Manuscript database

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

Ribosomal proteins L5 and L11 co-operatively inactivate c-Myc via RNA-induced silencing complex.

PubMed

Liao, J-M; Zhou, X; Gatignol, A; Lu, H

2014-10-09

Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.

RNAi therapeutics and applications of microRNAs in cancer treatment.

PubMed

Uchino, Keita; Ochiya, Takahiro; Takeshita, Fumitaka

2013-06-01

RNA interference-based therapies are proving to be powerful tools for combating various diseases, including cancer. Scientists are researching the development of safe and efficient systems for the delivery of small RNA molecules, which are extremely fragile in serum, to target organs and cells in the human body. A dozen pre-clinical and clinical trials have been under way over the past few years involving biodegradable nanoparticles, lipids, chemical modification and conjugation. On the other hand, microRNAs, which control the balance of cellular biological processes, have been studied as attractive therapeutic targets in cancer treatment. In this review, we provide an overview of RNA interference-based therapeutics in clinical trials and discuss the latest technology for the systemic delivery of nucleic acid drugs. Furthermore, we focus on dysregulated microRNAs in human cancer, which have progressed in pre-clinical trials as therapeutic targets, and describe a wide range of strategies to control the expression levels of endogenous microRNAs. Further development of RNA interference technologies and progression of clinical trials will contribute to the achievement of practical applications of nucleic acid drugs.

Space-time adaptive decision feedback neural receivers with data selection for high-data-rate users in DS-CDMA systems.

PubMed

de Lamare, Rodrigo C; Sampaio-Neto, Raimundo

2008-11-01

A space-time adaptive decision feedback (DF) receiver using recurrent neural networks (RNNs) is proposed for joint equalization and interference suppression in direct-sequence code-division multiple-access (DS-CDMA) systems equipped with antenna arrays. The proposed receiver structure employs dynamically driven RNNs in the feedforward section for equalization and multiaccess interference (MAI) suppression and a finite impulse response (FIR) linear filter in the feedback section for performing interference cancellation. A data selective gradient algorithm, based upon the set-membership (SM) design framework, is proposed for the estimation of the coefficients of RNN structures and is applied to the estimation of the parameters of the proposed neural receiver structure. Simulation results show that the proposed techniques achieve significant performance gains over existing schemes.

Representations of temporal information in short-term memory: Are they modality-specific?

PubMed

Bratzke, Daniel; Quinn, Katrina R; Ulrich, Rolf; Bausenhart, Karin M

2016-10-01

Rattat and Picard (2012) reported that the coding of temporal information in short-term memory is modality-specific, that is, temporal information received via the visual (auditory) modality is stored as a visual (auditory) code. This conclusion was supported by modality-specific interference effects on visual and auditory duration discrimination, which were induced by secondary tasks (visual tracking or articulatory suppression), presented during a retention interval. The present study assessed the stability of these modality-specific interference effects. Our study did not replicate the selective interference pattern but rather indicated that articulatory suppression not only impairs short-term memory for auditory but also for visual durations. This result pattern supports a crossmodal or an abstract view of temporal encoding. Copyright © 2016 Elsevier B.V. All rights reserved.

Interleukin-10-induced gene expression and suppressive function are selectively modulated by the PI3K-Akt-GSK3 pathway

PubMed Central

Antoniv, Taras T; Ivashkiv, Lionel B

2011-01-01

Interleukin-10 (IL-10) is an immunosuppressive cytokine that inhibits inflammatory gene expression. Phosphatidylinositol 3-kinase (PI3K) -mediated signalling regulates inflammatory responses and can induce IL-10 production, but a role for PI3K signalling in cellular responses to IL-10 is not known. In this study we investigated the involvement of the PI3K-Akt-GSK3 signalling pathway in IL-10-induced gene expression and IL-10-mediated suppression of Toll-like receptor-induced gene expression in primary human macrophages. A combination of loss and gain of function approaches using kinase inhibitors, expression of constitutively active Akt, and RNA interference in primary human macrophages showed that expression of a subset of IL-10-inducible genes was dependent on PI3K-Akt signalling. The effects of PI3K-Akt signalling on IL-10 responses were mediated at least in part by glycogen synthase kinase 3 (GSK3). In accordance with a functional role for PI3K pathways in contributing to the suppressive actions of IL-10, PI3K signalling augmented IL-10-mediated inhibition of lipopolysaccharide-induced IL-1, IL-8 and cyclo-oxygenase-2 expression. The PI3K signalling selectively modulated IL-10 responses, as it was not required for inhibition of tumour necrosis factor expression or for induction of certain IL-10-inducible genes such as SOCS3. These findings identify a new mechanism by which PI3K-mediated signalling can suppress inflammation by regulating IL-10-mediated gene induction and anti-inflammatory function. PMID:21255011

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.

Language-motor interference reflected in MEG beta oscillations.

PubMed

Klepp, Anne; Niccolai, Valentina; Buccino, Giovanni; Schnitzler, Alfons; Biermann-Ruben, Katja

2015-04-01

The involvement of the brain's motor system in action-related language processing can lead to overt interference with simultaneous action execution. The aim of the current study was to find evidence for this behavioural interference effect and to investigate its neurophysiological correlates using oscillatory MEG analysis. Subjects performed a semantic decision task on single action verbs, describing actions executed with the hands or the feet, and abstract verbs. Right hand button press responses were given for concrete verbs only. Therefore, longer response latencies for hand compared to foot verbs should reflect interference. We found interference effects to depend on verb imageability: overall response latencies for hand verbs did not differ significantly from foot verbs. However, imageability interacted with effector: while response latencies to hand and foot verbs with low imageability were equally fast, those for highly imageable hand verbs were longer than for highly imageable foot verbs. The difference is reflected in motor-related MEG beta band power suppression, which was weaker for highly imageable hand verbs compared with highly imageable foot verbs. This provides a putative neuronal mechanism for language-motor interference where the involvement of cortical hand motor areas in hand verb processing interacts with the typical beta suppression seen before movements. We found that the facilitatory effect of higher imageability on action verb processing time is perturbed when verb and motor response relate to the same body part. Importantly, this effect is accompanied by neurophysiological effects in beta band oscillations. The attenuated power suppression around the time of movement, reflecting decreased cortical excitability, seems to result from motor simulation during action-related language processing. This is in line with embodied cognition theories. Copyright © 2015. Published by Elsevier Inc.

Harnessing RNA interference to develop neonatal therapies: from Nobel Prize winning discovery to proof of concept clinical trials.

PubMed

DeVincenzo, John P

2009-10-01

A revolution in the understanding of RNA biological processing and control is leading to revolutionary new concepts in human therapeutics. It has become increasingly clear that the so called "non-coding RNA" exerts specific and profound functional control on regulation of protein production and indeed controls the expression of all genes. Harnessing this naturally-occurring RNA-mediated regulation of protein production has immense human therapeutic potential. These processes are collectively known as RNA interference (RNAi). RNAi is a recently discovered, naturally-occurring intracellular process that regulates gene expression through the silencing of specific mRNAs. Methods of harnessing this natural pathway are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small inhibitory RNAs (siRNA). siRNAs are being chemically modified to acquire drug-like properties. Numerous recent high profile publications have provided proofs of concept that RNA interference may be useful therapeutically. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. This makes this approach very attractive for developing therapies targeting orphan diseases including neonatal diseases. Theoretically, any disease that can be ameliorated through knockdown of any endogenous or exogenous protein is a potential therapeutic target for RNAi-based therapeutics. Lung diseases are particularly attractive targets for RNAi therapeutics since the affected cells' location increases their accessibility to topical administration of siRNA, for example by aerosol. Respiratory viral infections and chronic lung disease are examples of such diseases. RNAi therapeutics have been shown to be active against RSV, parainfluenza and human metapneumoviruses in vitro and in vivo resulting in profound antiviral effects. The first proof of concept test of efficacy of an RNAi-based therapeutic in man has been initiated. A discussion of the science behind RNA interference is followed by a presentation of the potential practical issues in applying this technology to neonatal respiratory viral diseases. RNAi may offer new strategies for the treatment of a variety of orphan diseases including neonatal diseases, RSV infections, and other respiratory viruses.

Adaptive suppression of power line interference in ultra-low field magnetic resonance imaging in an unshielded environment.

PubMed

Huang, Xiaolei; Dong, Hui; Qiu, Yang; Li, Bo; Tao, Quan; Zhang, Yi; Krause, Hans-Joachim; Offenhäusser, Andreas; Xie, Xiaoming

2018-01-01

Power-line harmonic interference and fixed-frequency noise peaks may cause stripe-artifacts in ultra-low field (ULF) magnetic resonance imaging (MRI) in an unshielded environment and in a conductively shielded room. In this paper we describe an adaptive suppression method to eliminate these artifacts in MRI images. This technique utilizes spatial correlation of the interference from different positions, and is realized by subtracting the outputs of the reference channel(s) from those of the signal channel(s) using wavelet analysis and the least squares method. The adaptive suppression method is first implemented to remove the image artifacts in simulation. We then experimentally demonstrate the feasibility of this technique by adding three orthogonal superconducting quantum interference device (SQUID) magnetometers as reference channels to compensate the output of one 2nd-order gradiometer. The experimental results show great improvement in the imaging quality in both 1D and 2D MRI images at two common imaging frequencies, 1.3 kHz and 4.8 kHz. At both frequencies, the effective compensation bandwidth is as high as 2 kHz. Furthermore, we examine the longitudinal relaxation times of the same sample before and after compensation, and show that the MRI properties of the sample did not change after applying adaptive suppression. This technique can effectively increase the imaging bandwidth and be applied to ULF MRI detected by either SQUIDs or Faraday coil in both an unshielded environment and a conductively shielded room. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel functions for the RNA-binding protein ETR-1 in Caenorhabditis elegans reproduction and engulfment of germline apoptotic cell corpses

PubMed Central

Boateng, Ruby; Nguyen, Ken C.Q.; Hall, David H.; Golden, Andy; Allen, Anna K.

2017-01-01

RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the somatic tissue and the germ line to ensure wild-type reproductive levels. Additionally, the ability of ETR-1 depletion to suppress the published WEE-1.3-depletion infertility phenotype is dependent on ETR-1 being reduced in the soma. Within the germline of etr-1(RNAi) hermaphrodite animals, we observe a decrease in average oocyte size and an increase in the number of germline apoptotic cell corpses as evident by an increased number of CED-1::GFP and acridine orange positive apoptotic germ cells. Transmission Electron Microscopy (TEM) studies confirm the significant increase in apoptotic cells in ETR-1-depleted animals, and reveal a failure of the somatic gonadal sheath cells to properly engulf dying germ cells in etr-1(RNAi) animals. Through investigation of an established engulfment pathway in C. elegans, we demonstrate that co-depletion of CED-1 and ETR-1 suppresses both the reduced fecundity and the increase in the number of apoptotic cell corpses observed in etr-1(RNAi) animals. Combined, this data identifies a novel role for ETR-1 in hermaphrodite gametogenesis and in the process of engulfment of germline apoptotic cell corpses. PMID:28648844

The impact of nevirapine- versus protease inhibitor-based regimens on virological markers of HIV-1 persistence during seemingly suppressive ART.

PubMed

Kiselinova, Maja; Anna, Maria; Malatinkova, Eva; Vervish, Karen; Beloukas, Apostolos; Messiaen, Peter; Bonczkowski, Pawel; Trypsteen, Wim; Callens, Steven; Verhofstede, Chris; De Spiegelaere, Ward; Vandekerckhove, Linos

2014-01-01

The source and significance of residual plasma HIV-1 RNA detection during suppressive ART remain controversial. It has been proposed that nevirapine (NVP)-based regimens achieve a greater HIV-1 RNA suppression than regimens containing a protease inhibitor (PI). The aim of this study was to compare the effect of receiving NVP- vs PI-based ART on the virological markers of HIV persistence in peripheral blood. The study population comprised 161 HIV-1 infected patients receiving either NVP-based (n=81) or PI-based (n=80) ART and showing a HIV-1 RNA load stably suppressed <40 copies/mL for median of 5.2 years (IQR 2.2-8.0). Residual viraemia was detected by real-time PCR with 50% and 95% detection thresholds of 1 and 3 HIV-1 RNA copies/mL, respectively. Cell-associated (CA) unspliced HIV-1 RNA, total HIV-1 DNA and 2 LTR circles were quantified in peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. Groups were compared by standard non-parametric tests; factors associated with HIV-1 detection were analyzed by univariate regression analysis and generalized linear models (SPSS® V22 and Rstudio). Plasma HIV-1 RNA was detected in 37/81 (45.7%) and 47/80 (58.8%) subjects on NVP- and PI-based ART, with median (IQR) levels of 5 (3-6) and 5 (3-8) copies/mL, respectively. HIV-1 RNA detection was associated with shorter duration of suppressive ART regardless of treatment arm (p=0.007), and lower CD4 nadir (p=0.015). HIV-1 DNA levels were median 282 (120-484) and 213 (87-494) copies/106 PBMCs in the two groups respectively, and were lowest (<100 copies/106 PBMCs) in subjects with lower plasma HIV-1 RNA (p=0.049), CA unspliced HIV-1 RNA (p=0.0001), 2 LTR circles (p=0.005) and pre-ART HIV-1 RNA load (p=0.0001). In this comprehensive characterization of patients on long-term suppressive ART, we did not observe evidence for a greater suppressive activity of NVP-based over PI-based therapy on plasma and intracellular markers of virus persistence. Overall excellent correlation was observed between the markers, allowing the identification of a subset of treated patients with low HIV-1 expression as an important cohort for future HIV cure studies.

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27.

PubMed

Sang, Dong-Ping; Li, Ru-Jun; Lan, Qing

2014-06-01

Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide (TMZ) in vitro. Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27. TMZ (200 or 400 μmol/L) alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin (30 μmol/L), TMZ (100 μmol/L) significantly inhibited the cell viability, and the inhibition of TMZ (200 and 400 μmol/L) was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression. Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.

Short-interfering RNAs Induce Retinal Degeneration via TLR3 and IRF3

PubMed Central

Kleinman, Mark E; Kaneko, Hiroki; Cho, Won Gil; Dridi, Sami; Fowler, Benjamin J; Blandford, Alexander D; Albuquerque, Romulo JC; Hirano, Yoshio; Terasaki, Hiroko; Kondo, Mineo; Fujita, Takashi; Ambati, Balamurali K; Tarallo, Valeria; Gelfand, Bradley D; Bogdanovich, Sasha; Baffi, Judit Z; Ambati, Jayakrishna

2012-01-01

The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these “naked” siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide. PMID:21988875

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects.

PubMed

Nguyen, Minh K; Jeon, Oju; Dang, Phuong N; Huynh, Cong T; Varghai, Davood; Riazi, Hooman; McMillan, Alexandra; Herberg, Samuel; Alsberg, Eben

2018-06-06

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

Doi, Nobutaka; New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd.; Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutuallymore » complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.« less

Silencing expression of UO-44 (CUZD1) using small interfering RNA sensitizes human ovarian cancer cells to cisplatin in vitro.

PubMed

Leong, C T C; Ong, C K; Tay, S K; Huynh, H

2007-02-08

Ovarian cancer is currently the second leading cause of gynecological malignancy and cisplatin or cisplatin-based regimens have been the standard of care for the treatment of advance epithelial ovarian cancers. However, the efficacy of cisplatin treatment is often limited by the development of drug resistance either through the inhibition of apoptotic genes or activation of antiapoptotic genes. We have previously reported the overexpression of human UO-44 (HuUO-44) in ovarian cancers and the HuUO-44 antisera markedly inhibited NIH-OVCAR3 ovarian cancer cell attachment and proliferation (Oncogene 23: 5707-5718, 2004). In the present study, we observed through the cancer cell line profiling array that the expression of HuUO-44 was suppressed in the ovarian cancer cell line (SKOV-3) after treatment with several chemotherapeutic drugs. Similarly, this suppression in HuUO-44 expression was also correlated to the cisplatin sensitivity in two other ovarian cancer cell lines NIH-OVCAR3 and OV-90 in a dose-dependent manner. To elucidate the function of HuUO-44 in cisplatin chemoresistance in ovarian cancer cell, small interfering RNAs (siRNAs) were employed to mediate HuUO-44 silencing in ovarian cancer cell line, NIH-OVCAR3. HuUO-44 RNA interference (RNAi) resulted in the inhibition of cell growth and proliferation. Importantly, HuUO-44 RNAi significantly increased sensitivity of NIH-OVCAR3 to cytotoxic stress induced by cisplatin (P<0.01). Strikingly, we have also demonstrated that overexpression of HuUO-44 significantly conferred cisplatin resistance in NIH-OVCAR3 cells (P<0.05). Taken together, UO-44 is involved in conferring cisplatin resistance; the described HuUO-44-specific siRNA oligonucleotides that can potently silence HuUO-44 gene expression may prove to be valuable pretreatment targets for antitumor therapy or other pathological conditions that involves aberrant HuUO-44 expression.

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

PubMed Central

Lo, Pang-Kuo; Lee, Ji Shin; Sukumar, Saraswati

2011-01-01

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G1 arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G1 arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1. PMID:21964066

Biochemical and Structural Studies of RNA Modification and Repair

ERIC Educational Resources Information Center

Chan, Chio Mui

2009-01-01

RNA modification, RNA interference, and RNA repair are important events in the cell. This thesis presents three projects related to these three fields. By using both biochemical and structural methods, we characterized enzymatic activities of pseudouridine synthase TruD, solved the structure of "A. aeolicus" GidA, and reconstituted a novel…

Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus.

PubMed

Hasiów-Jaroszewska, Beata; Minicka, Julia; Zarzyńska-Nowak, Aleksandra; Budzyńska, Daria; Elena, Santiago F

2018-05-02

Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species. Copyright © 2018 Elsevier B.V. All rights reserved.

RNA Interference for improving the Outcome of Islet Transplantation

PubMed Central

Li, Feng; Mahato, Ram I

2010-01-01

Islet transplantation has the potential to cure type 1 diabetes. Despite recent therapeutic success, it is still not common because a large number of transpanted islets get damaged by multiple challenges including instant blood mediated inflammatory reaction, hypoxia/reperfusion injury, inflammatory cytokines, and immune rejection. RNA interference (RNAi) is an novel strategy to selectively degrade target mRNA. The use of RNAi technologies to downregulate the expression of harmful genes has the potential to improve the outcome of islet transplantation. The aim of this review is to gain a thorough understanding of biological obstacles to islet transplantation and discuss how to overcome these barriers using different RNAi technologies. This eventually will help improve islet survival and function post transplantaion. Chemically synthesized small interferring RNA (siRNA), vector based short haripin RNA (shRNA), and their critical design elements (such as sequences, promoters, backbone) are discussed. The application of combinatorial RNAi in islet transplantation is also discussed. Last but not the least, several delivery strategies for enhanced gene silencing are discussed, including chemical modification of siRNA, complex formation, bioconjugation, and viral vectors. PMID:21156190

Differentiating RNA from DNA by a molecular fluorescent probe based on the "door-bolt" mechanism biomaterials.

PubMed

Yao, Qichao; Li, Haidong; Xian, Liman; Xu, Feng; Xia, Jing; Fan, Jiangli; Du, Jianjun; Wang, Jingyun; Peng, Xiaojun

2018-09-01

Although excellent florescent probes have been developed for DNA, good probes for RNA remain lacking. The shortage of reported and commercial RNA probes is attributable to their severe interference from DNA. As DNA and RNA have similar structures but different functions, it has been an imperative challenge to develop RNA probes that differentiate from DNA. In this study, an NIR fluorescent probe, NBE, is described, which contains a bulky julolidine group that can fit in a spacious RNA pocket and emit intense fluorescence. However, NBE has no response to DNA, as it cannot intercalate into the double strands or even in the DNA minor groove. The sensing mechanism is similar to the effect of a door-bolt. NBE shows excellent performance in RNA sensing (outstanding photostability, high selectivity and fast response), whether in aqueous buffers, fixed cells or living cells. These findings might provide not only a potential imaging tool but also a new design strategy for the recognition of RNA while avoiding interference from DNA. Copyright © 2018 Elsevier Ltd. All rights reserved.

Measles virus induces persistent infection by autoregulation of viral replication.

PubMed

Doi, Tomomitsu; Kwon, Hyun-Jeong; Honda, Tomoyuki; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

2016-11-24

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

Activation of activator protein 2 alpha by aspirin alleviates atherosclerotic plaque growth and instability in vivo

PubMed Central

Yang, Jing-Jing; Li, Peng; Wang, Fu; Liang, Wen-Jing; Ma, Hui; Chen, Yuan; Ma, Zhi-Min; Li, Quan-Zhong; Peng, Qi-Sheng; Zhang, Yun; Wang, Shuang-Xi

2016-01-01

Aims Aspirin has been used for the secondary prevention and treatment of cardiovascular disease for several decades. We investigated the roles of transcriptional factor activator protein 2α (AP-2α) in the beneficial effects of aspirin in the growth and vulnerability of atherosclerotic plaque. Methods and Results In mice deficient of apolipoprotein E (Apoe-/-), aspirin (20, 50 mg/kg/day) suppressed the progression of atherosclerosis in aortic roots and increased the plaque stability in carotid atherosclerotic plaques induced by collar-placement. In vivo lentivirus-mediated RNA interference of AP-2α reversed the inhibitory effects of aspirin on atherosclerosis in Apoe-/- mice. Mechanically, aspirin increased AP-2α phosphorylation and its activity, upregulated IkBα mRNA and protein levels, and reduced oxidative stress in cultured vascular smooth muscle cells. Furthermore, deficiency of AP-2α completely abolished aspirin-induced upregulation of IkBα levels and inhibition of oxidative stress in Apoe-/- mice. Clinically, conventional doses of aspirin increased AP-2α phosphorylation and IkBα protein expression in humans subjects. Conclusion Aspirin activates AP-2α to upregulate IkBα gene expression, resulting in attenuations of plaque development and instability in atherosclerosis. PMID:27391154

Reactive and proactive interference control in adults with autism spectrum disorder across the lifespan.

PubMed

Lever, Anne G; Ridderinkhof, K Richard; Marsman, Maarten; Geurts, Hilde M

2017-02-01

As a large heterogeneity is observed across studies on interference control in autism spectrum disorder (ASD), research may benefit from the use of a cognitive framework that models specific processes underlying reactive and proactive control of interference. Reactive control refers to the expression and suppression of responses and proactive control refers to the adjustment of response to previous situations. We administered a Simon conflict task in 2 independent adult samples (IQ >80) and applied distributional analyses to examine temporal dynamics of interference control in ASD. Along comparable interference effects in both reactive and proactive control, young men (n = 23, 18-36 years) diagnosed with ASD made as many fast errors on conflict trials as neurotypical controls (n = 19) and showed similar suppression on slow responses (Study 1). However, over the adult life span (19-79 years), individuals with ASD (n = 118) made fewer fast errors on conflict trials, and had overall slower and more accurate responses than controls (n = 160; Study 2). These results converge to the idea that individuals with ASD adopt a more cautious response bias over the adult life span, which is not yet observed among young adults. Our findings suggest that it is fruitful to distinguish different processes involved in interference control and contribute to an increased understanding of interference control mechanisms in adults with ASD. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Ribonucleic acid interference knockdown of interleukin 6 attenuates cold-induced hypertension.

PubMed

Crosswhite, Patrick; Sun, Zhongjie

2010-06-01

The purpose of this study was to determine the role of the proinflammatory cytokine interleukin (IL) 6 in cold-induced hypertension. Four groups of male Sprague-Dawley rats were used (6 rats per group). After blood pressure was stabilized, 3 groups received intravenous delivery of adenoassociated virus carrying IL-6 small hairpin RNA (shRNA), adenoassociated virus carrying scrambled shRNA, and PBS, respectively, before exposure to a cold environment (5 degrees C). The last group received PBS and was kept at room temperature (25 degrees C, warm) as a control. Adenoassociated virus delivery of IL-6 shRNA significantly attenuated cold-induced elevation of systolic blood pressure and kept it at the control level for < or =7 weeks (length of the study). Chronic exposure to cold upregulated IL-6 expression in aorta, heart, and kidneys and increased macrophage and T-cell infiltration in kidneys, suggesting that cold exposure increases inflammation. IL-6 shRNA delivery abolished the cold-induced upregulation of IL-6, indicating effective silence of IL-6. Interestingly, RNA interference knockdown of IL-6 prevented cold-induced inflammation, as evidenced by a complete inhibition of tumor necrosis factor-alpha expression and leukocyte infiltration by IL-6 shRNA. RNA interference knockdown of IL-6 significantly decreased the cold-induced increase in vascular superoxide production. It is noted that IL-6 shRNA abolished the cold-induced increase in collagen deposition in the heart, suggesting that inflammation is involved in cold-induced cardiac remodeling. Cold exposure caused glomerular collapses, which could be prevented by knockdown of IL-6, suggesting an important role of inflammation in cold-induced renal damage. In conclusion, cold exposure increased IL-6 expression and inflammation, which play critical roles in the pathogenesis of cold-induced hypertension and cardiac and renal damage.

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

PubMed

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

2014-04-01

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

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

New Genetics

MedlinePlus

... Century-Old Evolutionary Puzzle Computing Genetics Model Organisms RNA Interference The New Genetics is a science education ... the basics of DNA and its molecular cousin RNA, and new directions in genetic research. The New ...

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

PubMed

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

2014-03-01

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

Chemical Ligation Reactions of Oligonucleotides for Biological and Medicinal Applications.

PubMed

Abe, Hiroshi; Kimura, Yasuaki

2018-01-01

Chemical ligation of oligonucleotides (ONs) is the key reaction for various ON-based technologies. We have tried to solve the problems of RNA interference (RNAi) technology by applying ON chemical ligation to RNAi. We designed a new RNAi system, called intracellular buildup RNAi (IBR-RNAi), where the RNA fragments are built up into active small-interference RNA (siRNA) in cells through a chemical ligation reaction. Using the phosphorothioate and iodoacetyl groups as reactive functional groups for the ligation, we achieved RNAi effects without inducing immune responses. Additionally, we developed a new chemical ligation for IBR-RNAi, which affords a more native-like structure in the ligated product. The new ligation method should be useful not only for IBR-RNAi but also for the chemical synthesis of biofunctional ONs.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2009-11-01

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

MicroRNA-22 and microRNA-140 suppress NF-{kappa}B activity by regulating the expression of NF-{kappa}B coactivators

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

Takata, Akemi; Otsuka, Motoyuki, E-mail: otsukamo-tky@umin.ac.jp; Kojima, Kentaro

2011-08-12

Highlights: {yields} miRNAs were screened for their ability to regulate NF-{kappa}B activity. {yields} miRNA-22 and miRNA-140-3p suppress NF-{kappa}B activity by regulating coactivators. {yields} miRNA-22 targets nuclear receptor coactivator 1 (NCOA1). {yields} miRNA-140-3p targets nuclear receptor-interacting protein 1 (NRIP1). -- Abstract: Nuclear factor {kappa}B (NF-{kappa}B) is a transcription factor that regulates a set of genes that are critical to many biological phenomena, including liver tumorigenesis. To identify microRNAs (miRNAs) that regulate NF-{kappa}B activity in the liver, we screened 60 miRNAs expressed in hepatocytes for their ability to modulate NF-{kappa}B activity. We found that miRNA-22 and miRNA-140-3p significantly suppressed NF-{kappa}B activity bymore » regulating the expression of nuclear receptor coactivator 1 (NCOA1) and nuclear receptor-interacting protein 1 (NRIP1), both of which are NF-{kappa}B coactivators. Our results provide new information about the roles of miRNAs in the regulation of NF-{kappa}B activity.« less

RNA interference of cytochrome P450 CYP6F subfamily genes affects susceptibility to different insecticides in Locusta migratoria.

PubMed

Guo, Yanqiong; Wu, Haihua; Zhang, Xueyao; Ma, Enbo; Guo, Yaping; Zhu, Kun Yan; Zhang, Jianzhen

2016-11-01

Many insect cytochrome P450s (CYPs) play critical roles in detoxification of insecticides. The CYP6 family is unique to the class Insecta, and its biochemical function has essentially been associated with the metabolism of xenobiotics. In this study, we sequenced and characterised the full-length cDNAs of five CYP genes from Locusta migratoria, a highly destructive agricultural pest worldwide. The five genes were predominantly expressed in brain, guts, fat bodies or Malpighian tubules. CYP6FE1, CYP6FF1 and CYP6FG1 were expressed at higher levels in fourth-instar nymphs than in other developmental stages. CYPFD2 is specifically expressed in adults, whereas CYP6FD1, CYP6FD2 and CYP6FE1 showed significantly lower expression in eggs than in other developmental stages. Deltamethrin suppressed CYP6FD1 expression in third-instar nymphs and upregulated the expression level of CYP6FD2, CYP6FF1 and CYP6FG1 at the dose of LD 10 . Efficient RNA interference-mediated gene silencing was established for four of the five CYP genes. Silencing of CYP6FF1 increased the nymphal mortality from 23 to 50% in response to deltamethrin. Silencing of CYP6FD2 and CYP6FE1 increased the nymphal mortality from 32 to 72 and 66%, respectively, to carbaryl. Three of the four CYP6F subfamily genes in L. migratoria were associated with the detoxification of deltamethrin or carbaryl. The role of CYPs in insecticide detoxification appears to be both gene and insecticide specific. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Letrozole regulates actin cytoskeleton polymerization dynamics in a SRC-1 dependent manner in the hippocampus of mice.

PubMed

Zhao, Yangang; Yu, Yanlan; Zhang, Yuanyuan; He, Li; Qiu, Linli; Zhao, Jikai; Liu, Mengying; Zhang, Jiqiang

2017-03-01

In the hippocampus, local estrogens (E 2 ) derived from testosterone that is catalyzed by aromatase play important roles in the regulation of hippocampal neural plasticity, but the underlying mechanisms remain unclear. The actin cytoskeleton contributes greatly to hippocampal synaptic plasticity; however, whether it is regulated by local E 2 and the related mechanisms remain to be elucidated. In this study, we first examined the postnatal developmental profiles of hippocampal aromatase and specific proteins responsible for actin cytoskeleton dynamics. Then we used aromatase inhibitor letrozole (LET) to block local E 2 synthesis and examined the changes of these proteins and steroid receptor coactivator-1 (SRC-1), the predominant coactivator for steroid nuclear receptors. Finally, SRC-1 specific RNA interference was used to examine the effects of SRC-1 on the expression of these actin remodeling proteins. The results showed a V-type profile for aromatase and increased profiles for actin cytoskeleton proteins in both male and female hippocampus without obvious sex differences. LET treatment dramatically decreased the F-actin/G-actin ratio, the expression of Rictor, phospho-AKT (ser473), Profilin-1, phospho-Cofilin (Ser3), and SRC-1 in a dose-dependent manner. In vitro studies demonstrated that LET induced downregulation of these proteins could be reversed by E 2 , and E 2 induced increase of these proteins were significantly suppressed by SRC-1 shRNA interference. These results for the first time clearly demonstrated that local E 2 inhibition could induce aberrant actin polymerization; they also showed an important role of SRC-1 in the mediation of local E 2 action on hippocampal synaptic plasticity by regulation of actin cytoskeleton dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor panobinostat and FLT3-specific inhibitors.

PubMed

Pietschmann, Kristin; Bolck, Hella Anna; Buchwald, Marc; Spielberg, Steffi; Polzer, Harald; Spiekermann, Karsten; Bug, Gesine; Heinzel, Thorsten; Böhmer, Frank-Dietmar; Krämer, Oliver H

2012-11-01

Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. ©2012 AACR.

Reduced 64Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer.

PubMed

Cai, Huawei; Wu, Jiu-sheng; Muzik, Otto; Hsieh, Jer-Tsong; Lee, Robert J; Peng, Fangyu

2014-04-01

Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01). Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.

LncRNA NEAT1 promotes autophagy in MPTP-induced Parkinson's disease through stabilizing PINK1 protein.

PubMed

Yan, Wang; Chen, Zhao-Ying; Chen, Jia-Qi; Chen, Hui-Min

2018-02-19

Long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) was found to be closely related to the pathological changes in brain and nervous system. However, the role of NEAT1 and its potential mechanism in Parkinson's disease (PD) largely remain uncharacterized. In this study, PD mouse model was established by intraperitoneal injection of MPTP. The numbers of TH + neurons, NEAT1 expression and the level of PINK1, LC3-II, LC3-I protein were assessed in PD mice. SH-SY5Y cells were treated with MPP + as PD cell model. RNA pull-down assay was used to identify the interaction between NEAT1 and PINK1 in vitro. The endogenous expression of NEAT1 was modified by lentiviral vector carrying interference sequence for NEAT1 in vivo. The numbers of TH + neurons significantly decreased in PD mice compared with the control. The expressions of NEAT1, PINK1 protein and LC3-II/LC3-I level were increased by MPTP in vitro and in vivo. Moreover, NEAT1 positively regulated the protein level of PINK1 through inhibition of PINK1 protein degradation. And NEAT1 mediated the effects of MPP + on SH-SY5Y cells through stabilization of PINK1 protein. The results of in vivo experiments revealed that NEAT1 knockdown could effectively suppress MPTP-induced autophagy in vivo that alleviated dopaminergic neuronal injury. LncRNA NEAT1 promoted the MPTP-induced autophagy in PD through stabilization of PINK1 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Language Learning and Control in Monolinguals and Bilinguals

ERIC Educational Resources Information Center

Bartolotti, James; Marian, Viorica

2012-01-01

Parallel language activation in bilinguals leads to competition between languages. Experience managing this interference may aid novel language learning by improving the ability to suppress competition from known languages. To investigate the effect of bilingualism on the ability to control native-language interference, monolinguals and bilinguals…

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference.

PubMed

Zhang, Wanna; Liu, Bing; Lu, Yanhui; Liang, Gemei

2017-04-01

Salivary enzymes of many piercing-sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3-4) and adults (PG3-5), using siRNA injection-based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA-treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3-4 and PG3-5 siRNA-treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury. © 2017 Wiley Periodicals, Inc.

Drinking a hot blood meal elicits a protective heat shock response in mosquitoes.

PubMed

Benoit, Joshua B; Lopez-Martinez, Giancarlo; Patrick, Kevin R; Phillips, Zachary P; Krause, Tyler B; Denlinger, David L

2011-05-10

The mosquito's body temperature increases dramatically when it takes a blood meal from a warm-blooded, vertebrate host. By using the yellow fever mosquito, Aedes aegypti, we demonstrate that this boost in temperature following a blood meal prompts the synthesis of heat shock protein 70 (Hsp70). This response, elicited by the temperature of the blood meal, is most robust in the mosquito's midgut. When RNA interference is used to suppress expression of hsp70, protein digestion of the blood meal is impaired, leading to production of fewer eggs. We propose that Hsp70 protects the mosquito midgut from the temperature stress incurred by drinking a hot blood meal. Similar increases in hsp70 were documented immediately after blood feeding in two other mosquitoes (Culex pipiens and Anopheles gambiae) and the bed bug, Cimex lectularius, suggesting that this is a common protective response in blood-feeding arthropods.

Progress Toward In Vivo Use of siRNAs-II

PubMed Central

Rettig, Garrett R; Behlke, Mark A

2012-01-01

RNA interference (RNAi) has been extensively employed for in vivo research since its use was first demonstrated in mammalian cells 10 years ago. Design rules have improved, and it is now routinely possible to obtain reagents that suppress expression of any gene desired. At the same time, increased understanding of the molecular basis of unwanted side effects has led to the development of chemical modification strategies that mitigate these concerns. Delivery remains the single greatest hurdle to widespread adoption of in vivo RNAi methods. However, exciting advances have been made and new delivery systems under development may help to overcome these barriers. This review discusses advances in RNAi biochemistry and biology that impact in vivo use and provides an overview of select publications that demonstrate interesting applications of these principles. Emphasis is placed on work with synthetic, small interfering RNAs (siRNAs) published since the first installment of this review which appeared in 2006. PMID:22186795

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

MEK1-independent activation of MAPK and MEK1-dependent activation of p70 S6 kinase by stem cell factor (SCF) in ovarian cancer cells

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

Liu, Lian, E-mail: tounao@126.com; Institute of Immunology, School of Medicine, Shandong University, Jinan 250012; Zhang, Xin

We discovered a stem cell factor (SCF)-triggered, MEK1-independent, and PI3K-dependent MAPK activation pathway in the Kit-expressing ovarian cancer cell line HEY. When we knocked down MEK1 with RNA interference (RNAi) to study the function of MEK1 on the proliferation and survival of ovarian cancer cells, we found that impaired cell growth still occurred after MEK1 expression had been suppressed, although MAPK activation remained intact. This suggests that there is MEK1-independent activation of MAPK in the SCF-induced ovarian cancer cell growth process, and that MEK1 still plays a crucial role in maintaining the malignant properties of ovarian cancer cells even whenmore » it fails to activate MAPK as expected.« less

The Plant Polyester Cutin: Biosynthesis, Structure, and Biological Roles.

PubMed

Fich, Eric A; Segerson, Nicholas A; Rose, Jocelyn K C

2016-04-29

Cutin, a polyester composed mostly of oxygenated fatty acids, serves as the framework of the plant cuticle. The same types of cutin monomers occur across most plant lineages, although some evolutionary trends are evident. Additionally, cutins from some species have monomer profiles that are characteristic of the related polymer suberin. Compositional differences likely have profound structural consequences, but little is known about cutin's molecular organization and architectural heterogeneity. Its biological importance is suggested by the wide variety of associated mutants and gene-silencing lines that show a disruption of cuticular integrity, giving rise to numerous physiological and developmental abnormalities. Mapping and characterization of these mutants, along with suppression of gene paralogs through RNA interference, have revealed much of the biosynthetic pathway and several regulatory factors; however, the mechanisms of cutin polymerization and its interactions with other cuticle and cell wall components are only now beginning to be resolved.

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma irradiation

DTIC Science & Technology

2011-10-01

release of L11 from the nucleolus to the nucleoplasm, where it binds to c-Myc protein, and to the cytoplasm, where it binds to c-myc mRNA. We also found...rRNA and ribosomal proteins (RPs), rRNA processing, and the as- sembly of the mature ribosome subunits in the nucleolus fol- lowed by their transport...from the nucleolus or from intact ribosomes to suppress MDM2 (68). However, whether L11 suppresses c-Myc in response to ribosomal stress is not known

Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

PubMed

Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

2015-01-30

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNA interference in the clinic: challenges and future directions

PubMed Central

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

2011-01-01

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

Domain-Generality of Timing-Based Serial Order Processes in Short-Term Memory: New Insights from Musical and Verbal Domains

PubMed Central

Kowialiewski, Benjamin; Majerus, Steve

2016-01-01

Several models in the verbal domain of short-term memory (STM) consider a dissociation between item and order processing. This view is supported by data demonstrating that different types of time-based interference have a greater effect on memory for the order of to-be-remembered items than on memory for the items themselves. The present study investigated the domain-generality of the item versus serial order dissociation by comparing the differential effects of time-based interfering tasks, such as rhythmic interference and articulatory suppression, on item and order processing in verbal and musical STM domains. In Experiment 1, participants had to maintain sequences of verbal or musical information in STM, followed by a probe sequence, this under different conditions of interference (no-interference, rhythmic interference, articulatory suppression). They were required to decide whether all items of the probe list matched those of the memory list (item condition) or whether the order of the items in the probe sequence matched the order in the memory list (order condition). In Experiment 2, participants performed a serial order probe recognition task for verbal and musical sequences ensuring sequential maintenance processes, under no-interference or rhythmic interference conditions. For Experiment 1, serial order recognition was not significantly more impacted by interfering tasks than was item recognition, this for both verbal and musical domains. For Experiment 2, we observed selective interference of the rhythmic interference condition on both musical and verbal order STM tasks. Overall, the results suggest a similar and selective sensitivity to time-based interference for serial order STM in verbal and musical domains, but only when the STM tasks ensure sequential maintenance processes. PMID:27992565

Domain-Generality of Timing-Based Serial Order Processes in Short-Term Memory: New Insights from Musical and Verbal Domains.

PubMed

Gorin, Simon; Kowialiewski, Benjamin; Majerus, Steve

2016-01-01

Several models in the verbal domain of short-term memory (STM) consider a dissociation between item and order processing. This view is supported by data demonstrating that different types of time-based interference have a greater effect on memory for the order of to-be-remembered items than on memory for the items themselves. The present study investigated the domain-generality of the item versus serial order dissociation by comparing the differential effects of time-based interfering tasks, such as rhythmic interference and articulatory suppression, on item and order processing in verbal and musical STM domains. In Experiment 1, participants had to maintain sequences of verbal or musical information in STM, followed by a probe sequence, this under different conditions of interference (no-interference, rhythmic interference, articulatory suppression). They were required to decide whether all items of the probe list matched those of the memory list (item condition) or whether the order of the items in the probe sequence matched the order in the memory list (order condition). In Experiment 2, participants performed a serial order probe recognition task for verbal and musical sequences ensuring sequential maintenance processes, under no-interference or rhythmic interference conditions. For Experiment 1, serial order recognition was not significantly more impacted by interfering tasks than was item recognition, this for both verbal and musical domains. For Experiment 2, we observed selective interference of the rhythmic interference condition on both musical and verbal order STM tasks. Overall, the results suggest a similar and selective sensitivity to time-based interference for serial order STM in verbal and musical domains, but only when the STM tasks ensure sequential maintenance processes.

A new strategy in the treatment of chemoresistant lung adenocarcinoma via specific siRNA transfection of SRF, E2F1, Survivin, HIF and STAT3.

PubMed

Stoleriu, Mircea Gabriel; Steger, Volker; Mustafi, Migdat; Michaelis, Martin; Cinatl, Jindrich; Schneider, Wilke; Nolte, Andrea; Kurz, Julia; Wendel, Hans Peter; Schlensak, Christian; Walker, Tobias

2014-11-01

According to the actual treatment strategies of lung cancer, the current therapeutic regimen is an individualized, multidisciplinary concept. The development of chemoresistance in the last decade represents the most important obstacle to an effective treatment. In our study, we examined a new therapeutic alternative in the treatment of multiresistant lung adenocarcinoma via siRNA-specific transfection of six crucial molecules involved in lung carcinogenesis [serum response factor(SFR), E2F1, Survivin, hypoxia inducible factor1 (HIF1), HIF2 and signal transducer and activator of transcription (STAT3)]. Three chemoresistant A549 adenocarcinoma cells were cultured under standard conditions at 37°C and 5% CO2. The chemoresistance against Vinflunine, Vinorelbine and Methotrexate was induced artificially. The A549 cells were transfected for 2 h at 37°C with specific siRNA targeting SRF, E2F1, Survivin, HIF1, HIF2 and STAT3 in a non-viral manner. The efficiency of siRNA silencing was evaluated via quantitative real-time polymerase chain reaction, whereas the surviving cells after siRNA transfection as predictor factor for tumoural growth were analysed with a CASY cell counter 3 days after transfection. The response of the chemotherapeutic resistant adenocarcinoma cells after siRNA transfection was concentration-dependent at both 25 and 100 nM. The CASY analysis showed a very effective suppression of adenocarcinoma cells in Vinorelbine, Vinflunine and Methotrexate groups, with significantly better results in comparison with the control group. In our study, we emphasized that siRNA interference might represent a productive platform for further research in order to investigate whether a new regimen in the treatment of multiresistant non-small-cell lung cancer could be established in vivo in the context of a multimodal cancer therapy. © The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference

PubMed Central

Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

2015-01-01

RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells. PMID:25731667

Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference.

PubMed

Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

2015-03-03

RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity.

PubMed

Huang, Chung-Hao; Hsiao, Weng-Rong; Huang, Ching-Wen; Chen, Kuan-Chun; Lin, Shih-Shun; Chen, Tsung-Chi; Raja, Joseph A J; Wu, Hui-Wen; Yeh, Shyi-Dong

2015-01-01

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) ((109)KFTMHNQ(117)), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif ((397)IYFL(400)) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

PubMed Central

Huang, Chung-Hao; Hsiao, Weng-Rong; Huang, Ching-Wen; Chen, Kuan-Chun; Lin, Shih-Shun; Chen, Tsung-Chi; Raja, Joseph A. J.; Wu, Hui-Wen; Yeh, Shyi-Dong

2015-01-01

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQ117), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism. PMID:25993336

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Suppression of emission rates improves sonar performance by flying bats.

PubMed

Adams, Amanda M; Davis, Kaylee; Smotherman, Michael

2017-01-31

Echolocating bats face the challenge of actively sensing their environment through their own emissions, while also hearing calls and echoes of nearby conspecifics. How bats mitigate interference is a long-standing question that has both ecological and technological implications, as biosonar systems continue to outperform man-made sonar systems in noisy, cluttered environments. We recently showed that perched bats decreased calling rates in groups, displaying a behavioral strategy resembling the back-off algorithms used in artificial communication networks to optimize information throughput at the group level. We tested whether free-tailed bats (Tadarida brasiliensis) would employ such a coordinated strategy while performing challenging flight maneuvers, and report here that bats navigating obstacles lowered emission rates when hearing artificial playback of another bat's calls. We measured the impact of acoustic interference on navigation performance and show that the calculated reductions in interference rates are sufficient to reduce interference and improve obstacle avoidance. When bats flew in pairs, each bat responded to the presence of the other as an obstacle by increasing emissions, but hearing the sonar emissions of the nearby bat partially suppressed this response. This behavior supports social cohesion by providing a key mechanism for minimizing mutual interference.

Suppression of emission rates improves sonar performance by flying bats

PubMed Central

Adams, Amanda M.; Davis, Kaylee; Smotherman, Michael

2017-01-01

Echolocating bats face the challenge of actively sensing their environment through their own emissions, while also hearing calls and echoes of nearby conspecifics. How bats mitigate interference is a long-standing question that has both ecological and technological implications, as biosonar systems continue to outperform man-made sonar systems in noisy, cluttered environments. We recently showed that perched bats decreased calling rates in groups, displaying a behavioral strategy resembling the back-off algorithms used in artificial communication networks to optimize information throughput at the group level. We tested whether free-tailed bats (Tadarida brasiliensis) would employ such a coordinated strategy while performing challenging flight maneuvers, and report here that bats navigating obstacles lowered emission rates when hearing artificial playback of another bat’s calls. We measured the impact of acoustic interference on navigation performance and show that the calculated reductions in interference rates are sufficient to reduce interference and improve obstacle avoidance. When bats flew in pairs, each bat responded to the presence of the other as an obstacle by increasing emissions, but hearing the sonar emissions of the nearby bat partially suppressed this response. This behavior supports social cohesion by providing a key mechanism for minimizing mutual interference. PMID:28139707

Modulation of alpha oscillations is required for the suppression of semantic interference.

PubMed

Melnik, Natalia; Mapelli, Igor; Özkurt, Tolga Esat

2017-10-01

Recent findings on alpha band oscillations suggest their important role in memory consolidation and suppression of external distractors such as environmental noise. However, less attention was given to the phenomenon of internal distracting information being solely inherent to the stimuli content. Human memory may be prone to internal distractions caused by semantic relatedness between the meaning of words (e.g., atom, neutron, nucleus, etc.) to be encoded, i.e., semantic interference. Our study investigates the brain oscillatory dynamics behind the semantic interference phenomenon, whose possible outcome is known as false memories. In this direction, Deese-Roediger-McDermott word lists were appropriated for a modified Sternberg paradigm in auditory modality. Participants received semantically related and unrelated word lists via headphones while EEG data were acquired. Semantic interference triggered the false memory rates to be higher than those of other types of memory errors. Analysis demonstrated that the upper part of alpha band (∼10-12Hz) power decreases on parieto-occipital channels in the retention interval, prior to the probe item for semantically related condition. Our study elucidates the oscillatory mechanisms behind semantic interference by relying on alpha functional inhibition theory. Copyright © 2017 Elsevier Inc. All rights reserved.

Perceptual load-dependent neural correlates of distractor interference inhibition.

PubMed

Xu, Jiansong; Monterosso, John; Kober, Hedy; Balodis, Iris M; Potenza, Marc N

2011-01-18

The load theory of selective attention hypothesizes that distractor interference is suppressed after perceptual processing (i.e., in the later stage of central processing) at low perceptual load of the central task, but in the early stage of perceptual processing at high perceptual load. Consistently, studies on the neural correlates of attention have found a smaller distractor-related activation in the sensory cortex at high relative to low perceptual load. However, it is not clear whether the distractor-related activation in brain regions linked to later stages of central processing (e.g., in the frontostriatal circuits) is also smaller at high rather than low perceptual load, as might be predicted based on the load theory. We studied 24 healthy participants using functional magnetic resonance imaging (fMRI) during a visual target identification task with two perceptual loads (low vs. high). Participants showed distractor-related increases in activation in the midbrain, striatum, occipital and medial and lateral prefrontal cortices at low load, but distractor-related decreases in activation in the midbrain ventral tegmental area and substantia nigra (VTA/SN), striatum, thalamus, and extensive sensory cortices at high load. Multiple levels of central processing involving midbrain and frontostriatal circuits participate in suppressing distractor interference at either low or high perceptual load. For suppressing distractor interference, the processing of sensory inputs in both early and late stages of central processing are enhanced at low load but inhibited at high load.

Multimodality Imaging of RNA Interference

PubMed Central

Nayak, Tapas R.; Krasteva, Lazura K.; Cai, Weibo

2013-01-01

The discovery of small interfering RNAs (siRNAs) and their potential to knock down virtually any gene of interest has ushered in a new era of RNA interference (RNAi). Clinical use of RNAi faces severe limitations due to inefficiency delivery of siRNA or short hairpin RNA (shRNA). Many molecular imaging techniques have been adopted in RNAi-related research for evaluation of siRNA/shRNA delivery, biodistribution, pharmacokinetics, and the therapeutic effect. In this review article, we summarize the current status of in vivo imaging of RNAi. The molecular imaging techniques that have been employed include bioluminescence/fluorescence imaging, magnetic resonance imaging/spectroscopy, positron emission tomography, single-photon emission computed tomography, and various combinations of these techniques. Further development of non-invasive imaging strategies for RNAi, not only focusing on the delivery of siRNA/shRNA but also the therapeutic efficacy, is critical for future clinical translation. Rigorous validation will be needed to confirm that biodistribution of the carrier is correlated with that of siRNA/shRNA, since imaging only detects the label (e.g. radioisotopes) but not the gene or carrier themselves. It is also essential to develop multimodality imaging approaches for realizing the full potential of therapeutic RNAi, as no single imaging modality may be sufficient to simultaneously monitor both the gene delivery and silencing effect of RNAi. PMID:23745567

Engineering functional inorganic-organic hybrid systems: advances in siRNA therapeutics.

PubMed

Shen, Jianliang; Zhang, Wei; Qi, Ruogu; Mao, Zong-Wan; Shen, Haifa

2018-03-21

Cancer treatment still faces a lot of obstacles such as tumor heterogeneity, drug resistance and systemic toxicities. Beyond the traditional treatment modalities, exploitation of RNA interference (RNAi) as an emerging approach has immense potential for the treatment of various gene-caused diseases including cancer. The last decade has witnessed enormous research and achievements focused on RNAi biotechnology. However, delivery of small interference RNA (siRNA) remains a key challenge in the development of clinical RNAi therapeutics. Indeed, functional nanomaterials play an important role in siRNA delivery, which could overcome a wide range of sequential physiological and biological obstacles. Nanomaterial-formulated siRNA systems have potential applications in protection of siRNA from degradation, improving the accumulation in the target tissues, enhancing the siRNA therapy and reducing the side effects. In this review, we explore and summarize the role of functional inorganic-organic hybrid systems involved in the siRNA therapeutic advancements. Additionally, we gather the surface engineering strategies of hybrid systems to optimize for siRNA delivery. Major progress in the field of inorganic-organic hybrid platforms including metallic/non-metallic cores modified with organic shells or further fabrication as the vectors for siRNA delivery is discussed to give credit to the interdisciplinary cooperation between chemistry, pharmacy, biology and medicine.

Parameters on plant absortion of double-stranded Ribonucleic acid, dsRNA

USDA-ARS?s Scientific Manuscript database

Efficient absorption of double-stranded Ribonucleic acid, dsRNA, into citrus is critical for effective psyllid management by RNA interference, RNAi. Parameters which might affect absorption into citrus trees and subsequent ingestion by Asian citrus psyllid were evaluated. Age of leaves, variety of c...

SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

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

Kubo, Takanori, E-mail: kubo-t@yasuda-u.ac.jp; Yanagihara, Kazuyoshi; Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045

2012-10-05

Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, suchmore » as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.« less

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

PubMed

Haga, K; Lemp, N A; Logg, C R; Nagashima, J; Faure-Kumar, E; Gomez, G G; Kruse, C A; Mendez, R; Stripecke, R; Kasahara, N; Kasahara, N A; Cicciarelli, J C

2006-12-01

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

PubMed Central

Harris, M E; Pace, N R

1995-01-01

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs. PMID:7585250

Molecular mechanisms influencing efficiency of RNA interference in insects.

PubMed

Cooper, Anastasia M W; Silver, Kristopher; Jianzhen, Zhang; Park, Yoonseong; Zhu, Kun Yan

2018-06-21

RNA interference (RNAi) is an endogenous, sequence-specific gene silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi-based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double-stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. The recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small-interfering RNA/double-stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.