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Sample records for multi-target antisense approach

  1. A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

    PubMed Central

    Fortin, Marylène; D'Anjou, Hélène; Higgins, Marie-Ève; Gougeon, Jasmine; Aubé, Paméla; Moktefi, Kamel; Mouissi, Sonia; Séguin, Serge; Séguin, Rosanne; Renzi, Paolo M; Paquet, Luc; Ferrari, Nicolay

    2009-01-01

    Background Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo. Methods Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs. Results In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation

  2. Multi-Target Approach to Metastatic Adrenal Cell Carcinoma.

    PubMed

    Wahab, Norasyikin A; Zainudin, Suehazlyn; AbAziz, Aini; Mustafa, Norlaila; Sukor, Norlela; Kamaruddin, Nor Azmi

    2016-09-01

    Adrenal cell carcinoma is a rare tumor and more than 70% of patients present with advanced stages. Adrenal cell carcinoma is an aggressive tumor with a poor prognosis. Surgical intervention is the gold standard treatment and mitotane is the only drug approved for the treatment of adrenal cell carcinoma. Until recently in 2012, the etoposide, doxorubicin, cisplatin plus mitotane are approved as first-line therapy based on response rate and progression-free survival. This case illustrates a case of advanced adrenal cell carcinoma in a young girl who presented with huge adrenal mass with inferior vena cava thrombosis and pulmonary embolism. Multi-approach of therapy was used to control the tumor size and metastasis. Therefore, it may prolong her survival rate for up to 5 years and 4 months. PMID:27631184

  3. PMHT Approach for Multi-Target Multi-Sensor Sonar Tracking in Clutter.

    PubMed

    Li, Xiaohua; Li, Yaan; Yu, Jing; Chen, Xiao; Dai, Miao

    2015-01-01

    Multi-sensor sonar tracking has many advantages, such as the potential to reduce the overall measurement uncertainty and the possibility to hide the receiver. However, the use of multi-target multi-sensor sonar tracking is challenging because of the complexity of the underwater environment, especially the low target detection probability and extremely large number of false alarms caused by reverberation. In this work, to solve the problem of multi-target multi-sensor sonar tracking in the presence of clutter, a novel probabilistic multi-hypothesis tracker (PMHT) approach based on the extended Kalman filter (EKF) and unscented Kalman filter (UKF) is proposed. The PMHT can efficiently handle the unknown measurements-to-targets and measurements-to-transmitters data association ambiguity. The EKF and UKF are used to deal with the high degree of nonlinearity in the measurement model. The simulation results show that the proposed algorithm can improve the target tracking performance in a cluttered environment greatly, and its computational load is low. PMID:26561817

  4. PMHT Approach for Multi-Target Multi-Sensor Sonar Tracking in Clutter.

    PubMed

    Li, Xiaohua; Li, Yaan; Yu, Jing; Chen, Xiao; Dai, Miao

    2015-11-06

    Multi-sensor sonar tracking has many advantages, such as the potential to reduce the overall measurement uncertainty and the possibility to hide the receiver. However, the use of multi-target multi-sensor sonar tracking is challenging because of the complexity of the underwater environment, especially the low target detection probability and extremely large number of false alarms caused by reverberation. In this work, to solve the problem of multi-target multi-sensor sonar tracking in the presence of clutter, a novel probabilistic multi-hypothesis tracker (PMHT) approach based on the extended Kalman filter (EKF) and unscented Kalman filter (UKF) is proposed. The PMHT can efficiently handle the unknown measurements-to-targets and measurements-to-transmitters data association ambiguity. The EKF and UKF are used to deal with the high degree of nonlinearity in the measurement model. The simulation results show that the proposed algorithm can improve the target tracking performance in a cluttered environment greatly, and its computational load is low.

  5. PMHT Approach for Multi-Target Multi-Sensor Sonar Tracking in Clutter

    PubMed Central

    Li, Xiaohua; Li, Yaan; Yu, Jing; Chen, Xiao; Dai, Miao

    2015-01-01

    Multi-sensor sonar tracking has many advantages, such as the potential to reduce the overall measurement uncertainty and the possibility to hide the receiver. However, the use of multi-target multi-sensor sonar tracking is challenging because of the complexity of the underwater environment, especially the low target detection probability and extremely large number of false alarms caused by reverberation. In this work, to solve the problem of multi-target multi-sensor sonar tracking in the presence of clutter, a novel probabilistic multi-hypothesis tracker (PMHT) approach based on the extended Kalman filter (EKF) and unscented Kalman filter (UKF) is proposed. The PMHT can efficiently handle the unknown measurements-to-targets and measurements-to-transmitters data association ambiguity. The EKF and UKF are used to deal with the high degree of nonlinearity in the measurement model. The simulation results show that the proposed algorithm can improve the target tracking performance in a cluttered environment greatly, and its computational load is low. PMID:26561817

  6. Systems biology approaches and tools for analysis of interactomes and multi-target drugs.

    PubMed

    Schrattenholz, André; Groebe, Karlfried; Soskic, Vukic

    2010-01-01

    diseases" remains a most pressing medical need. Currently, a change of paradigm can be observed with regard to a new interest in agents that modulate multiple targets simultaneously, essentially "dirty drugs." Targeting cellular function as a system rather than on the level of the single target, significantly increases the size of the drugable proteome and is expected to introduce novel classes of multi-target drugs with fewer adverse effects and toxicity. Multiple target approaches have recently been used to design medications against atherosclerosis, cancer, depression, psychosis and neurodegenerative diseases. A focussed approach towards "systemic" drugs will certainly require the development of novel computational and mathematical concepts for appropriate modelling of complex data. But the key is the extraction of relevant molecular information from biological systems by implementing rigid statistical procedures to differential proteomic analytics.

  7. A multi-targeted approach to suppress tumor-promoting inflammation.

    PubMed

    Samadi, Abbas K; Bilsland, Alan; Georgakilas, Alexandros G; Amedei, Amedeo; Amin, Amr; Bishayee, Anupam; Azmi, Asfar S; Lokeshwar, Bal L; Grue, Brendan; Panis, Carolina; Boosani, Chandra S; Poudyal, Deepak; Stafforini, Diana M; Bhakta, Dipita; Niccolai, Elena; Guha, Gunjan; Vasantha Rupasinghe, H P; Fujii, Hiromasa; Honoki, Kanya; Mehta, Kapil; Aquilano, Katia; Lowe, Leroy; Hofseth, Lorne J; Ricciardiello, Luigi; Ciriolo, Maria Rosa; Singh, Neetu; Whelan, Richard L; Chaturvedi, Rupesh; Ashraf, S Salman; Shantha Kumara, H M C; Nowsheen, Somaira; Mohammed, Sulma I; Keith, W Nicol; Helferich, William G; Yang, Xujuan

    2015-12-01

    Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes.

  8. Finding structures with specific properties in complex configurational spaces using multi-target inverse band structure approach

    NASA Astrophysics Data System (ADS)

    Piquini, Paulo; Zunger, Alex

    2009-03-01

    The conventional strategy to look for materials with desired properties is to use physical intuition to select some candidates among an enormous number of possibilities.Apart the very special cases, the solutions to these search problems are far from obvious. The inverse band structure (IBS) approach, on the other hand, search for the desired electronic structures (instead of atomic configurations) from the beginning. Here we illustrate the power of this inverse approach by applying it to the simultaneous engineering of multi-target problems, which encompass huge configurational spaces: (i) the search of a specific band gap in the quaternary (In,Ga)(As,Sb) semiconductors(a) lattice-matched to InP and, (ii) the stacking sequence of (In,Ga)As/InP superlattices leading to band gaps and strains within the range suitable for thermophotovoltaic applications(b). [3pt] (a) P. Piquini, P.A. Graf, and A. Zunger, Phys. Rev. Lett. 100, 186403 (2008); [0pt] (b) P. Piquini and A. Zunger, Phys. Rev. B 78, 161302 (2008)

  9. Antisense Modulation of RNA Processing as a Therapeutic Approach in Cancer Therapy

    PubMed Central

    Spraggon, Lee

    2013-01-01

    Next-generation antisense technologies are re-emerging as viable and powerful approaches to the treatment of several genetic diseases. Similar strategies are also being applied to cancer therapy. Re-programming of the expression of endogenous oncogenic products to replace them with functional antagonists, by interfering with alternative splicing or polyadenylation, provides a promising novel approach to address acquired drug resistance and previously undruggable targets. PMID:25589899

  10. A Network-Based Data Integration Approach to Support Drug Repurposing and Multi-Target Therapies in Triple Negative Breast Cancer.

    PubMed

    Vitali, Francesca; Cohen, Laurie D; Demartini, Andrea; Amato, Angela; Eterno, Vincenzo; Zambelli, Alberto; Bellazzi, Riccardo

    2016-01-01

    The integration of data and knowledge from heterogeneous sources can be a key success factor in drug design, drug repurposing and multi-target therapies. In this context, biological networks provide a useful instrument to highlight the relationships and to model the phenomena underlying therapeutic action in cancer. In our work, we applied network-based modeling within a novel bioinformatics pipeline to identify promising multi-target drugs. Given a certain tumor type/subtype, we derive a disease-specific Protein-Protein Interaction (PPI) network by combining different data-bases and knowledge repositories. Next, the application of suitable graph-based algorithms allows selecting a set of potentially interesting combinations of drug targets. A list of drug candidates is then extracted by applying a recent data fusion approach based on matrix tri-factorization. Available knowledge about selected drugs mechanisms of action is finally exploited to identify the most promising candidates for planning in vitro studies. We applied this approach to the case of Triple Negative Breast Cancer (TNBC), a subtype of breast cancer whose biology is poorly understood and that lacks of specific molecular targets. Our "in-silico" findings have been confirmed by a number of in vitro experiments, whose results demonstrated the ability of the method to select candidates for drug repurposing. PMID:27632168

  11. A Network-Based Data Integration Approach to Support Drug Repurposing and Multi-Target Therapies in Triple Negative Breast Cancer

    PubMed Central

    Cohen, Laurie D.; Demartini, Andrea; Amato, Angela; Eterno, Vincenzo; Zambelli, Alberto; Bellazzi, Riccardo

    2016-01-01

    The integration of data and knowledge from heterogeneous sources can be a key success factor in drug design, drug repurposing and multi-target therapies. In this context, biological networks provide a useful instrument to highlight the relationships and to model the phenomena underlying therapeutic action in cancer. In our work, we applied network-based modeling within a novel bioinformatics pipeline to identify promising multi-target drugs. Given a certain tumor type/subtype, we derive a disease-specific Protein-Protein Interaction (PPI) network by combining different data-bases and knowledge repositories. Next, the application of suitable graph-based algorithms allows selecting a set of potentially interesting combinations of drug targets. A list of drug candidates is then extracted by applying a recent data fusion approach based on matrix tri-factorization. Available knowledge about selected drugs mechanisms of action is finally exploited to identify the most promising candidates for planning in vitro studies. We applied this approach to the case of Triple Negative Breast Cancer (TNBC), a subtype of breast cancer whose biology is poorly understood and that lacks of specific molecular targets. Our “in-silico” findings have been confirmed by a number of in vitro experiments, whose results demonstrated the ability of the method to select candidates for drug repurposing. PMID:27632168

  12. Multi-target parallel processing approach for gene-to-structure determination of the influenza polymerase PB2 subunit.

    PubMed

    Armour, Brianna L; Barnes, Steve R; Moen, Spencer O; Smith, Eric; Raymond, Amy C; Fairman, James W; Stewart, Lance J; Staker, Bart L; Begley, Darren W; Edwards, Thomas E; Lorimer, Donald D

    2013-01-01

    Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year (1). Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans (2). Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target. The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design. Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains. PMID:23851357

  13. Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

    PubMed Central

    Moen, Spencer O.; Smith, Eric; Raymond, Amy C.; Fairman, James W.; Stewart, Lance J.; Staker, Bart L.; Begley, Darren W.; Edwards, Thomas E.; Lorimer, Donald D.

    2013-01-01

    Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target. The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design. Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains. PMID:23851357

  14. Multi-Target Directed Drugs: A Modern Approach for Design of New Drugs for the treatment of Alzheimer's Disease.

    PubMed

    Dias, Kris Simone Tranches; Viegas, Claudio

    2014-05-01

    Alzheimer's disease (AD) is a complex neurodegenerative disorder with a multi-faceted pathogenesis. So far, the therapeutic paradigm "one-compound-one-target" has failed and despite enormous efforts to elucidate the pathophysiology of AD, the disease is still incurable. The multiple factors involved in AD include amyloid aggregation to form insoluble neurotoxic plaques of Aβ, hyperphosphorylation of tau protein, oxidative stress, calcium imbalance, mitochondrial dysfunction and deterioration of synaptic transmission. These factors together, accentuate changes in the CNS homeostasis, starting a complex process of interconnected physiological damage, leading to cognitive and memory impairment and neuronal death. A recent approach for the rational design of new drug candidates, also called multitarget-directed ligand (MTDL) approach, has gained increasing attention by many research groups, which have developed a variety of hybrid compounds acting simultaneously on diverse biological targets. This review aims to show some recent advances and examples of the exploitation of MTDL approach in the rational design of novel drug candidate prototypes for the treatment of AD.

  15. Antisense approaches for elucidating ranavirus gene function in an infected fish cell line.

    PubMed

    Whitley, D S; Sample, R C; Sinning, A R; Henegar, J; Chinchar, V G

    2011-09-01

    Viral virulence/immune evasion strategies and host anti-viral responses represent different sides of the continuing struggle between virus and host survival. To identify virus-encoding molecules whose function is to subvert or blunt host immune responses, we have adapted anti-sense approaches to knock down the expression of specific viral gene products. Our intention is to correlate knock down with loss of function and thus infer the role of a given viral gene. As a starting point in this process we have targeted several structural and catalytic genes using antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA). In proof of concept experiments we show the feasibility of this approach and describe recent work targeting five frog virus 3 genes. Our results indicate that both 46K and 32R, two immediate-early viral proteins, are essential for replication in vitro, and confirm earlier findings that the major capsid protein, the largest subunit of the viral homolog of RNA polymerase II, and the viral DNA methyltransferase are also essential for replication in cell culture.

  16. Identification of REST-regulated genes and pathways using a REST-targeted antisense approach.

    PubMed

    Sedaghat, Yalda; Bui, Huynh-Hoa; Mazur, Curt; Monia, Brett P

    2013-12-01

    The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is one of the first negative-acting transcriptional regulators implicated in vertebrate development thought to regulate hundreds of neuron-specific genes. However, its function in the adult system remains elusive. Here we employ second-generation antisense oligonucleotides (ASOs) to study the impact of rest-mediated suppression on gene expression. We demonstrate specific reductions in REST levels in vitro, and in vivo in mouse liver following treatment with ASOs, and we show that ASO mediated-REST suppression results in the elevation in expression of many neuronal genes including brain-derived neurotrophic factor, Synapsin1 (syn1) and β3-tubulin in BALB/c liver. Furthermore, we show the elevation of the affected proteins in plasma following ASO treatment. Finally, microarray analysis was applied to identify a broad range of genes modulated by REST suppression in mouse liver. Our findings suggest that REST may be an important target for neurodegenerative diseases like Huntington's disease, is also involved in the regulation of a broad range of additional cellular pathways, and that the antisense approach is a viable strategy for selectively modulating REST activity in vivo. PMID:24329414

  17. Multi-Target QSAR Approaches for Modeling Protein Inhibitors. Simultaneous Prediction of Activities Against Biomacromolecules Present in Gram-Negative Bacteria.

    PubMed

    Speck-Planche, Alejandro; Cordeiro, M N D S

    2015-01-01

    Drug discovery is aimed at finding therapeutic agents for the treatment of many diverse diseases and infections. However, this is a very slow an expensive process, and for this reason, in silico approaches are needed to rationalize the search for new molecular entities with desired biological profiles. Models focused on quantitative structure-activity relationships (QSAR) have constituted useful complementary tools in medicinal chemistry, allowing the virtual predictions of dissimilar pharmacological activities of compounds. In the last 10 years, multi-target (mt) QSAR models have been reported, representing great advances with respect to those models generated from classical approaches. Thus, mt- QSAR models can simultaneously predict activities against different biological targets (proteins, microorganisms, cell lines, etc.) by using large and heterogeneous datasets of chemicals. The present review is devoted to discuss the most promising mt-QSAR models, particularly those developed for the prediction of protein inhibitors. We also report the first multi-tasking QSAR (mtk-QSAR) model for simultaneous prediction of inhibitors against biomacromolecules (specifically proteins) present in Gram-negative bacteria. This model allowed us to consider both different proteins and multiple experimental conditions under which the inhibitory activities of the chemicals were determined. The mtk-QSAR model exhibited accuracies higher than 98% in both training and prediction sets, also displaying a very good performance in the classification of active and inactive cases that depended on the specific elements of the experimental conditions. The physicochemical interpretations of the molecular descriptors were also analyzed, providing important insights regarding the molecular patterns associated with the appearance/enhancement of the inhibitory potency. PMID:25961517

  18. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach.

    PubMed

    Bordoy, Antoni E; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  19. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach

    PubMed Central

    Bordoy, Antoni E.; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  20. A Multi-Target Approach toward the Development of Novel Candidates for Antidermatophytic Activity: Ultrastructural Evidence on α-Bisabolol-Treated Microsporum gypseum.

    PubMed

    Romagnoli, Carlo; Baldisserotto, Anna; Malisardi, Gemma; Vicentini, Chiara B; Mares, Donatella; Andreotti, Elisa; Vertuani, Silvia; Manfredini, Stefano

    2015-01-01

    Multi-target strategies are directed toward targets that are unrelated (or distantly related) and can create opportunities to address different pathologies. The antidermatophytic activities of nine natural skin lighteners: α-bisabolol, kojic acid, β-arbutin, azelaic acid, hydroquinone, nicotinamide, glycine, glutathione and ascorbyl tetraisopalmitate, were evaluated, in comparison with the known antifungal drug fluconazole, on nine dermatophytes responsible for the most common dermatomycoses: Microsporum gypseum, Microsporum canis, Trichophyton violaceum, Nannizzia cajetani, Trichophyton mentagrophytes, Epidermophyton floccosum, Arthroderma gypseum, Trichophyton rubrum and Trichophyton tonsurans. α-Bisabolol showed the best antifungal activity against all fungi and in particular; against M. gypseum. Further investigations were conducted on this fungus to evaluate the inhibition of spore germination and morphological changes induced by α-bisabolol by TEM. PMID:26132903

  1. A capillary based chemiluminscent multi-target immunoassay.

    PubMed

    Cao, Yuan-Cheng

    2015-05-01

    Renewed interest in capillary format immunoassays has lead to increasingly costly and complex approaches to preparation and readout. This study describes a simple multi-target method based on a capillary platform using horseradish peroxidase (HRP) labelled IgG to visualize an antibody antigen complex. When goat-anti-human IgG was employed as the probe and human IgG as target, the system allowed detection of target to less than 1 ng/mL using a standard detection approach. The capillaries were read visually or with a commercial grade CCD camera. Multi-target detection was demonstrated using a model system of rat-anti-mouse, goat-anti-human and mouse-anti-rat IgG. These probes were encoded to different locations in the capillary, providing a simple inexpensive approach to achieve multi-target assays.

  2. Cubature Information SMC-PHD for Multi-Target Tracking.

    PubMed

    Liu, Zhe; Wang, Zulin; Xu, Mai

    2016-01-01

    In multi-target tracking, the key problem lies in estimating the number and states of individual targets, in which the challenge is the time-varying multi-target numbers and states. Recently, several multi-target tracking approaches, based on the sequential Monte Carlo probability hypothesis density (SMC-PHD) filter, have been presented to solve such a problem. However, most of these approaches select the transition density as the importance sampling (IS) function, which is inefficient in a nonlinear scenario. To enhance the performance of the conventional SMC-PHD filter, we propose in this paper two approaches using the cubature information filter (CIF) for multi-target tracking. More specifically, we first apply the posterior intensity as the IS function. Then, we propose to utilize the CIF algorithm with a gating method to calculate the IS function, namely CISMC-PHD approach. Meanwhile, a fast implementation of the CISMC-PHD approach is proposed, which clusters the particles into several groups according to the Gaussian mixture components. With the constructed components, the IS function is approximated instead of particles. As a result, the computational complexity of the CISMC-PHD approach can be significantly reduced. The simulation results demonstrate the effectiveness of our approaches. PMID:27171088

  3. Cubature Information SMC-PHD for Multi-Target Tracking.

    PubMed

    Liu, Zhe; Wang, Zulin; Xu, Mai

    2016-05-09

    In multi-target tracking, the key problem lies in estimating the number and states of individual targets, in which the challenge is the time-varying multi-target numbers and states. Recently, several multi-target tracking approaches, based on the sequential Monte Carlo probability hypothesis density (SMC-PHD) filter, have been presented to solve such a problem. However, most of these approaches select the transition density as the importance sampling (IS) function, which is inefficient in a nonlinear scenario. To enhance the performance of the conventional SMC-PHD filter, we propose in this paper two approaches using the cubature information filter (CIF) for multi-target tracking. More specifically, we first apply the posterior intensity as the IS function. Then, we propose to utilize the CIF algorithm with a gating method to calculate the IS function, namely CISMC-PHD approach. Meanwhile, a fast implementation of the CISMC-PHD approach is proposed, which clusters the particles into several groups according to the Gaussian mixture components. With the constructed components, the IS function is approximated instead of particles. As a result, the computational complexity of the CISMC-PHD approach can be significantly reduced. The simulation results demonstrate the effectiveness of our approaches.

  4. Cubature Information SMC-PHD for Multi-Target Tracking

    PubMed Central

    Liu, Zhe; Wang, Zulin; Xu, Mai

    2016-01-01

    In multi-target tracking, the key problem lies in estimating the number and states of individual targets, in which the challenge is the time-varying multi-target numbers and states. Recently, several multi-target tracking approaches, based on the sequential Monte Carlo probability hypothesis density (SMC-PHD) filter, have been presented to solve such a problem. However, most of these approaches select the transition density as the importance sampling (IS) function, which is inefficient in a nonlinear scenario. To enhance the performance of the conventional SMC-PHD filter, we propose in this paper two approaches using the cubature information filter (CIF) for multi-target tracking. More specifically, we first apply the posterior intensity as the IS function. Then, we propose to utilize the CIF algorithm with a gating method to calculate the IS function, namely CISMC-PHD approach. Meanwhile, a fast implementation of the CISMC-PHD approach is proposed, which clusters the particles into several groups according to the Gaussian mixture components. With the constructed components, the IS function is approximated instead of particles. As a result, the computational complexity of the CISMC-PHD approach can be significantly reduced. The simulation results demonstrate the effectiveness of our approaches. PMID:27171088

  5. Multi-Target Directed Drugs: A Modern Approach for Design of New Drugs for the treatment of Alzheimer’s Disease

    PubMed Central

    Dias, Kris Simone Tranches; Viegas, Jr, Claudio

    2014-01-01

    Alzheimer’s disease (AD) is a complex neurodegenerative disorder with a multi-faceted pathogenesis. So far, the therapeutic paradigm “one-compound-one-target” has failed and despite enormous efforts to elucidate the pathophysiology of AD, the disease is still incurable. The multiple factors involved in AD include amyloid aggregation to form insoluble neurotoxic plaques of Aβ, hyperphosphorylation of tau protein, oxidative stress, calcium imbalance, mitochondrial dysfunction and deterioration of synaptic transmission. These factors together, accentuate changes in the CNS homeostasis, starting a complex process of interconnected physiological damage, leading to cognitive and memory impairment and neuronal death. A recent approach for the rational design of new drug candidates, also called multitarget-directed ligand (MTDL) approach, has gained increasing attention by many research groups, which have developed a variety of hybrid compounds acting simultaneously on diverse biological targets. This review aims to show some recent advances and examples of the exploitation of MTDL approach in the rational design of novel drug candidate prototypes for the treatment of AD. PMID:24851088

  6. Modelling ligand selectivity of serine proteases using integrative proteochemometric approaches improves model performance and allows the multi-target dependent interpretation of features.

    PubMed

    Ain, Qurrat U; Méndez-Lucio, Oscar; Ciriano, Isidro Cortés; Malliavin, Thérèse; van Westen, Gerard J P; Bender, Andreas

    2014-11-01

    Serine proteases, implicated in important physiological functions, have a high intra-family similarity, which leads to unwanted off-target effects of inhibitors with insufficient selectivity. However, the availability of sequence and structure data has now made it possible to develop approaches to design pharmacological agents that can discriminate successfully between their related binding sites. In this study, we have quantified the relationship between 12,625 distinct protease inhibitors and their bioactivity against 67 targets of the serine protease family (20,213 data points) in an integrative manner, using proteochemometric modelling (PCM). The benchmarking of 21 different target descriptors motivated the usage of specific binding pocket amino acid descriptors, which helped in the identification of active site residues and selective compound chemotypes affecting compound affinity and selectivity. PCM models performed better than alternative approaches (models trained using exclusively compound descriptors on all available data, QSAR) employed for comparison with R(2)/RMSE values of 0.64 ± 0.23/0.66 ± 0.20 vs. 0.35 ± 0.27/1.05 ± 0.27 log units, respectively. Moreover, the interpretation of the PCM model singled out various chemical substructures responsible for bioactivity and selectivity towards particular proteases (thrombin, trypsin and coagulation factor 10) in agreement with the literature. For instance, absence of a tertiary sulphonamide was identified to be responsible for decreased selective activity (by on average 0.27 ± 0.65 pChEMBL units) on FA10. Among the binding pocket residues, the amino acids (arginine, leucine and tyrosine) at positions 35, 39, 60, 93, 140 and 207 were observed as key contributing residues for selective affinity on these three targets.

  7. An in vivo and in silico approach to study cis-antisense: a short cut to higher order response

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen; Varanasi, Usha; Chatterjee, Anushree

    2014-03-01

    Antisense interactions are present in all domains of life. Typically sense, antisense RNA pairs originate from overlapping genes with convergent face to face promoters, and are speculated to be involved in gene regulation. Recent studies indicate the role of transcriptional interference (TI) in regulating expression of genes in convergent orientation. Modeling antisense, TI gene regulation mechanisms allows us to understand how organisms control gene expression. We present a modeling and experimental framework to understand convergent transcription that combines the effects of transcriptional interference and cis-antisense regulation. Our model shows that combining transcriptional interference and antisense RNA interaction adds multiple-levels of regulation which affords a highly tunable biological output, ranging from first order response to complex higher-order response. To study this system we created a library of experimental constructs with engineered TI and antisense interaction by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences to control expression of a set of fluorescent reporter proteins. Studying this gene expression mechanism allows for an understanding of higher order behavior of gene expression networks.

  8. Sequential measurement-driven multi-target Bayesian filter

    NASA Astrophysics Data System (ADS)

    Liu, Zong-xiang; Li, Li-juan; Xie, Wei-xin; Li, Liang-qun

    2015-12-01

    Bayesian filter is an efficient approach for multi-target tracking in the presence of clutter. Recently, considerable attention has been focused on probability hypothesis density (PHD) filter, which is an intensity approximation of the multi-target Bayesian filter. However, PHD filter is inapplicable to cases in which target detection probability is low. The use of this filter may result in a delay in data processing because it handles received measurements periodically, once every sampling period. To track multiple targets in the case of low detection probability and to handle received measurements in real time, we propose a sequential measurement-driven Bayesian filter. The proposed filter jointly propagates the marginal distributions and existence probabilities of each target in the filter recursion. We also present an implementation of the proposed filter for linear Gaussian models. Simulation results demonstrate that the proposed filter can more accurately track multiple targets than the Gaussian mixture PHD filter or cardinalized PHD filter.

  9. Targeting Cancer with Antisense Oligomers

    SciTech Connect

    Hnatowich, DJ

    2008-10-28

    With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their native and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes

  10. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

    PubMed Central

    Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé

    2015-01-01

    Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni. PMID:26452552

  11. The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium.

    PubMed

    Darsonval, Maud; Msadek, Tarek; Alexandre, Hervé; Grandvalet, Cosette

    2015-10-09

    Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.

  12. Mid-course multi-target tracking using continuous representation

    NASA Technical Reports Server (NTRS)

    Zak, Michail; Toomarian, Nikzad

    1991-01-01

    The thrust of this paper is to present a new approach to multi-target tracking for the mid-course stage of the Strategic Defense Initiative (SDI). This approach is based upon a continuum representation of a cluster of flying objects. We assume that the velocities of the flying objects can be embedded into a smooth velocity field. This assumption is based upon the impossibility of encounters in a high density cluster between the flying objects. Therefore, the problem is reduced to an identification of a moving continuum based upon consecutive time frame observations. In contradistinction to the previous approaches, here each target is considered as a center of a small continuous neighborhood subjected to a local-affine transformation, and therefore, the target trajectories do not mix. Obviously, their mixture in plane of sensor view is apparent. The approach is illustrated by an example.

  13. Random finite set multi-target trackers: stochastic geometry for space situational awareness

    NASA Astrophysics Data System (ADS)

    Vo, Ba-Ngu; Vo, Ba-Tuong

    2015-05-01

    This paper describes the recent development in the random finite set RFS paradigm in multi-target tracking. Over the last decade the Probability Hypothesis Density filter has become synonymous with the RFS approach. As result the PHD filter is often wrongly used as a performance benchmark for the RFS approach. Since there is a suite of RFS-based multi-target tracking algorithms, benchmarking tracking performance of the RFS approach by using the PHD filter, the cheapest of these, is misleading. Such benchmarking should be performed with more sophisticated RFS algorithms. In this paper we outline the high-performance RFS-based multi-target trackers such that the Generalized Labled Multi-Bernoulli filter, and a number of efficient approximations and discuss extensions and applications of these filters. Applications to space situational awareness are discussed.

  14. Upping the Antisense Ante.

    ERIC Educational Resources Information Center

    Weiss, Rick

    1991-01-01

    Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)

  15. Bayesian multi-target tracking and sequential object recognition

    NASA Astrophysics Data System (ADS)

    Armbruster, Walter

    2008-04-01

    The paper considers the following problem: given a 3D model of a reference target and a sequence of images of a 3D scene, identify the object in the scene most likely to be the reference target and determine its current pose. Finding the best match in each frame independently of previous decisions is not optimal, since past information is ignored. Our solution concept uses a novel Bayesian framework for multi target tracking and object recognition to define and sequentially update the probability that the reference target is any one of the tracked objects. The approach is applied to problems of automatic lock-on and missile guidance using a laser radar seeker. Field trials have resulted in high target hit probabilities despite low resolution imagery and temporarily highly occluded targets.

  16. A Particle Multi-Target Tracker for Superpositional Measurements Using Labeled Random Finite Sets

    NASA Astrophysics Data System (ADS)

    Papi, Francesco; Kim, Du Yong

    2015-08-01

    In this paper we present a general solution for multi-target tracking with superpositional measurements. Measurements that are functions of the sum of the contributions of the targets present in the surveillance area are called superpositional measurements. We base our modelling on Labeled Random Finite Set (RFS) in order to jointly estimate the number of targets and their trajectories. This modelling leads to a labeled version of Mahler's multi-target Bayes filter. However, a straightforward implementation of this tracker using Sequential Monte Carlo (SMC) methods is not feasible due to the difficulties of sampling in high dimensional spaces. We propose an efficient multi-target sampling strategy based on Superpositional Approximate CPHD (SA-CPHD) filter and the recently introduced Labeled Multi-Bernoulli (LMB) and Vo-Vo densities. The applicability of the proposed approach is verified through simulation in a challenging radar application with closely spaced targets and low signal-to-noise ratio.

  17. Diversification of antisense research and development: review of the Ringberg meeting, April 1994. Mechanisms of antisense-mediated gene silencing.

    PubMed

    Hawkins, J W; Nellen, W

    1994-01-01

    Antisense technology has established itself as a new and vibrant entrant into the discipline of molecular biology. As such, it has contributed to basic research by providing tools for the molecular dissection of diverse experimental systems. In applied research, antisense approaches have contributed to development of agricultural products (D. Grierson) now coming to market and to the design of a number of oligonucleotide drugs, now in clinical trials. However, few activities to date have focused on the study of antisense per se. Further, few conceptual perspectives have regarded antisense as an integral part of cellular function and genetic regulation. The Ringberg conference showcased a number of systems that would seem unrelated if we regard antisense as a superficial tool to be imposed on nature. On the other hand, if we want to begin to regard antisense as a field of its own with deeper biological and genetic rationales, the Ringberg meeting provided much tantalizing evidence to do so.

  18. Multi-target pharmacology: possibilities and limitations of the “skeleton key approach” from a medicinal chemist perspective

    PubMed Central

    Talevi, Alan

    2015-01-01

    Multi-target drugs have raised considerable interest in the last decade owing to their advantages in the treatment of complex diseases and health conditions linked to drug resistance issues. Prospective drug repositioning to treat comorbid conditions is an additional, overlooked application of multi-target ligands. While medicinal chemists usually rely on some version of the lock and key paradigm to design novel therapeutics, modern pharmacology recognizes that the mid- and long-term effects of a given drug on a biological system may depend not only on the specific ligand-target recognition events but also on the influence of the repeated administration of a drug on the cell gene signature. The design of multi-target agents usually imposes challenging restrictions on the topology or flexibility of the candidate drugs, which are briefly discussed in the present article. Finally, computational strategies to approach the identification of novel multi-target agents are overviewed. PMID:26441661

  19. System-level multi-target drug discovery from natural products with applications to cardiovascular diseases.

    PubMed

    Zheng, Chunli; Wang, Jinan; Liu, Jianling; Pei, Mengjie; Huang, Chao; Wang, Yonghua

    2014-08-01

    The term systems pharmacology describes a field of study that uses computational and experimental approaches to broaden the view of drug actions rooted in molecular interactions and advance the process of drug discovery. The aim of this work is to stick out the role that the systems pharmacology plays across the multi-target drug discovery from natural products for cardiovascular diseases (CVDs). Firstly, based on network pharmacology methods, we reconstructed the drug-target and target-target networks to determine the putative protein target set of multi-target drugs for CVDs treatment. Secondly, we reintegrated a compound dataset of natural products and then obtained a multi-target compounds subset by virtual-screening process. Thirdly, a drug-likeness evaluation was applied to find the ADME-favorable compounds in this subset. Finally, we conducted in vitro experiments to evaluate the reliability of the selected chemicals and targets. We found that four of the five randomly selected natural molecules can effectively act on the target set for CVDs, indicating the reasonability of our systems-based method. This strategy may serve as a new model for multi-target drug discovery of complex diseases.

  20. Making sense of antisense

    SciTech Connect

    Moffat, A.S.

    1991-08-02

    Out in the San Joaquin Valley of California grows a field of tomato plants that look like ordinary tomato plants. But these tomatoes are special - one of the first fruits of a new technology that may revolutionize not just commercial plant development but human medicine as well. The new technology uses novel RNAs, called antisense RNAs, to block the activity of specific genes. At first, researchers were mainly interested in antisense RNA as a tool for probing gene function. In the late 1970s, when the technology was first developed, molecular biologists didn't have a good way of mutating genes in the cells of higher organisms so that they could see what happens when the gene activity is lost. Antisense technology, in effect, provided a way of doing that. But the biotechnology industry soon recognized the immense practical potential of a technique that could be used to knock out the activity of bad genes. To make the tomato plants, for example, plant scientists used antisense RNAs to shut off the expression of the gene encoding an enzyme that makes tomatoes mushy, thereby yielding a product that may travel better and last longer on grocery shelves. Recent work by various labs suggests that it may be possible to design antisense compounds that inhibit the activity of viral genes or of the oncogenes thought to contribute to cancer development, without affecting normal cellular genes. That raises the possibility that the technology might aid in producing better, more selective drugs to treat viral diseases, including AIDS, and cancer.

  1. Acute Molecular Perturbation of Inducible Nitric Oxide Synthase with an Antisense Approach Enhances Neuronal Preservation and Functional Recovery after Contusive Spinal Cord Injury

    PubMed Central

    Maggio, Dominic M.; Chatzipanteli, Katina; Masters, Neil; Patel, Samik P.; Dietrich, W. Dalton

    2012-01-01

    Abstract Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation and oxidative stress produced during pathological conditions, including neurodegenerative diseases and central nervous system (CNS) injury. iNOS is responsible for the formation of high levels of nitric oxide (NO). The production of highly reactive and cytotoxic NO species, such as peroxynitrite, plays an important role in secondary tissue damage. We have previously demonstrated that acute administration of iNOS antisense oligonucleotides (ASOs) 3 h after moderate contusive spinal cord injury (SCI) potently inhibits iNOS-mediated increases in NO levels, leading to reduced blood–spinal cord barrier permeability, decreased neutrophil accumulation, and less neuronal cell death. In the current study we investigated if iNOS ASOs could also provide long-term (10-week) histological and behavioral improvements after moderate thoracic T8 contusive SCI. Adult rats were randomly assigned to three groups (n=10/group): SCI alone, SCI and mixed base control oligonucleotides (MBOs), or SCI and iNOS ASOs (200 nM). Oligonucleotides were administered by spinal superfusion 3 h after injury. Behavioral analysis (Basso-Beattie-Bresnahan [BBB] score and subscore) was employed weekly for 10 weeks post-SCI. Although animals treated with iNOS ASOs demonstrated no significant differences in BBB scores compared to controls, subscore analysis revealed a significant improvement in foot positioning, trunk stability, and tail clearance. Histologically, while no gross improvement in preserved white and gray matter was observed, greater numbers of surviving neurons were present adjacent to the lesion site in iNOS ASO-treated animals than controls. These results support the effectiveness of targeting iNOS acutely as a therapeutic approach after SCI. PMID:22708918

  2. FISST based method for multi-target tracking in the image plane of optical sensors.

    PubMed

    Xu, Yang; Xu, Hui; An, Wei; Xu, Dan

    2012-01-01

    A finite set statistics (FISST)-based method is proposed for multi-target tracking in the image plane of optical sensors. The method involves using signal amplitude information in probability hypothesis density (PHD) filter which is derived from FISST to improve multi-target tracking performance. The amplitude of signals generated by the optical sensor is modeled first, from which the amplitude likelihood ratio between target and clutter is derived. An alternative approach is adopted for the situations where the signal noise ratio (SNR) of target is unknown. Then the PHD recursion equations incorporated with signal information are derived and the Gaussian mixture (GM) implementation of this filter is given. Simulation results demonstrate that the proposed method achieves significantly better performance than the generic PHD filter. Moreover, our method has much lower computational complexity in the scenario with high SNR and dense clutter. PMID:22736984

  3. FISST Based Method for Multi-Target Tracking in the Image Plane of Optical Sensors

    PubMed Central

    Xu, Yang; Xu, Hui; An, Wei; Xu, Dan

    2012-01-01

    A finite set statistics (FISST)-based method is proposed for multi-target tracking in the image plane of optical sensors. The method involves using signal amplitude information in probability hypothesis density (PHD) filter which is derived from FISST to improve multi-target tracking performance. The amplitude of signals generated by the optical sensor is modeled first, from which the amplitude likelihood ratio between target and clutter is derived. An alternative approach is adopted for the situations where the signal noise ratio (SNR) of target is unknown. Then the PHD recursion equations incorporated with signal information are derived and the Gaussian mixture (GM) implementation of this filter is given. Simulation results demonstrate that the proposed method achieves significantly better performance than the generic PHD filter. Moreover, our method has much lower computational complexity in the scenario with high SNR and dense clutter. PMID:22736984

  4. FISST based method for multi-target tracking in the image plane of optical sensors.

    PubMed

    Xu, Yang; Xu, Hui; An, Wei; Xu, Dan

    2012-01-01

    A finite set statistics (FISST)-based method is proposed for multi-target tracking in the image plane of optical sensors. The method involves using signal amplitude information in probability hypothesis density (PHD) filter which is derived from FISST to improve multi-target tracking performance. The amplitude of signals generated by the optical sensor is modeled first, from which the amplitude likelihood ratio between target and clutter is derived. An alternative approach is adopted for the situations where the signal noise ratio (SNR) of target is unknown. Then the PHD recursion equations incorporated with signal information are derived and the Gaussian mixture (GM) implementation of this filter is given. Simulation results demonstrate that the proposed method achieves significantly better performance than the generic PHD filter. Moreover, our method has much lower computational complexity in the scenario with high SNR and dense clutter.

  5. [Control of gene expression by antisense nucleic acids].

    PubMed

    Lebleu, B; Clarenc, J P; Degols, G; Leonetti, J P; Milhaud, P

    1992-01-01

    The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.

  6. Multi-Target Tracking by Discrete-Continuous Energy Minimization.

    PubMed

    Milan, Anton; Schindler, Konrad; Roth, Stefan

    2016-10-01

    The task of tracking multiple targets is often addressed with the so-called tracking-by-detection paradigm, where the first step is to obtain a set of target hypotheses for each frame independently. Tracking can then be regarded as solving two separate, but tightly coupled problems. The first is to carry out data association, i.e., to determine the origin of each of the available observations. The second problem is to reconstruct the actual trajectories that describe the spatio-temporal motion pattern of each individual target. The former is inherently a discrete problem, while the latter should intuitively be modeled in continuous space. Having to deal with an unknown number of targets, complex dependencies, and physical constraints, both are challenging tasks on their own and thus most previous work focuses on one of these subproblems. Here, we present a multi-target tracking approach that explicitly models both tasks as minimization of a unified discrete-continuous energy function. Trajectory properties are captured through global label costs, a recent concept from multi-model fitting, which we introduce to tracking. Specifically, label costs describe physical properties of individual tracks, e.g., linear and angular dynamics, or entry and exit points. We further introduce pairwise label costs to describe mutual interactions between targets in order to avoid collisions. By choosing appropriate forms for the individual energy components, powerful discrete optimization techniques can be leveraged to address data association, while the shapes of individual trajectories are updated by gradient-based continuous energy minimization. The proposed method achieves state-of-the-art results on diverse benchmark sequences.

  7. Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms.

    PubMed

    Wang, H; Nan, L; Yu, D; Agrawal, S; Zhang, R

    2001-11-01

    The mouse double minute 2 (MDM2) oncogene has been suggested as a target for cancer therapy. It is amplified or overexpressed in many human cancers, including breast cancer, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer, ovarian cancer, osteosarcoma, and lymphoma. In the present study, we investigated the functions of MDM2 oncogene in the growth of breast cancer and the potential value of MDM2 as a drug target for cancer therapy by inhibiting MDM2 expression with a specific antisense antihuman-MDM2 oligonucleotide (oligo). The selected antisense mixed-backbone oligo was evaluated for its in vitro and in vivo antitumor activity in human breast cancer models: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. In MCF-7 cells, p53 and p21 levels were elevated, resulting from specific inhibition of MDM2 expression by the antisense oligo (AS). In MDA-MB-468 cells, after inhibition of MDM2 expression, p21 levels were elevated, although p53 levels remained unchanged. After i.p. administration of the antisense anti-MDM2 oligo, in vivo antitumor activity occurred in a dose-dependent manner in nude mice bearing MCF-7 or MDA-MB-468 xenografts. In both models, in vivo synergistically or additive therapeutic effects of MDM2 inhibition and the clinically used cancer chemotherapeutic agents irinotecan, 5-fluorouracil, and paclitaxel (Taxol) were observed. These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53-independent mechanisms. We speculate that MDM2 inhibitors, such as ASs, have a broad spectrum of antitumor activities in human breast cancers, regardless of p53 status. This study should provide a basis for future development of anti-MDM2 ASs as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.

  8. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    PubMed

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. PMID:26787513

  9. The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach

    PubMed Central

    Swiatkowska, Agata; Zydowicz, Paulina; Gorska, Agnieszka; Suchacka, Julia; Dutkiewicz, Mariola; Ciesiołka, Jerzy

    2015-01-01

    The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors. PMID:26513723

  10. Inferring multi-target QSAR models with taxonomy-based multi-task learning

    PubMed Central

    2013-01-01

    Background A plethora of studies indicate that the development of multi-target drugs is beneficial for complex diseases like cancer. Accurate QSAR models for each of the desired targets assist the optimization of a lead candidate by the prediction of affinity profiles. Often, the targets of a multi-target drug are sufficiently similar such that, in principle, knowledge can be transferred between the QSAR models to improve the model accuracy. In this study, we present two different multi-task algorithms from the field of transfer learning that can exploit the similarity between several targets to transfer knowledge between the target specific QSAR models. Results We evaluated the two methods on simulated data and a data set of 112 human kinases assembled from the public database ChEMBL. The relatedness between the kinase targets was derived from the taxonomy of the humane kinome. The experiments show that multi-task learning increases the performance compared to training separate models on both types of data given a sufficient similarity between the tasks. On the kinase data, the best multi-task approach improved the mean squared error of the QSAR models of 58 kinase targets. Conclusions Multi-task learning is a valuable approach for inferring multi-target QSAR models for lead optimization. The application of multi-task learning is most beneficial if knowledge can be transferred from a similar task with a lot of in-domain knowledge to a task with little in-domain knowledge. Furthermore, the benefit increases with a decreasing overlap between the chemical space spanned by the tasks. PMID:23842210

  11. Sense and antisense transcription are associated with distinct chromatin architectures across genes.

    PubMed

    Murray, Struan C; Haenni, Simon; Howe, Françoise S; Fischl, Harry; Chocian, Karolina; Nair, Anitha; Mellor, Jane

    2015-09-18

    Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

  12. An inexpensive multi-target carousel for PLD systems

    NASA Astrophysics Data System (ADS)

    Clark, J. H.; Weston, R. G.

    1996-05-01

    Pulsed laser deposition (PLD) of thin films is widely regarded as the best growth technique for the development of novel multilayer structures and devices; in particular, high-temperature superconducting (HTS) devices. To achieve this, it is essential to have the capability to deposit material from different targets sequentially in situ, namely without opening the deposition chamber. Here we present details of a multi-target carousel for multilayer depositions. It has been designed to allow target rotation and selection without any latching mechanisms or in-vacuum motors and is also suitable for use in automated systems. Additionally, it is robust, relatively inexpensive, compact, scalable and simple to build. Single-layer 0957-0233/7/5/014/img1 (YBCO) thin films grown using the multi-target carousel show no reduction in quality compared to ones grown using a single-target system. Moreover, the carousel has successfully been used to deposit Ag onto YBCO in situ to realize low-resistance YBCO - Ag - YBCO resistor structures; which would have been impossible with ex situ metallization. In addition, in situ homo-epitaxial MgO buffer layers on (100) MgO substrates prior to the deposition of YBCO have been investigated as a means of improving HTS film quality.

  13. Viral escape from antisense RNA.

    PubMed

    Bull, J J; Jacobson, A; Badgett, M R; Molineux, I J

    1998-05-01

    RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition. PMID:9643550

  14. Network Pharmacology Strategies Toward Multi-Target Anticancer Therapies: From Computational Models to Experimental Design Principles

    PubMed Central

    Tang, Jing; Aittokallio, Tero

    2014-01-01

    Polypharmacology has emerged as novel means in drug discovery for improving treatment response in clinical use. However, to really capitalize on the polypharmacological effects of drugs, there is a critical need to better model and understand how the complex interactions between drugs and their cellular targets contribute to drug efficacy and possible side effects. Network graphs provide a convenient modeling framework for dealing with the fact that most drugs act on cellular systems through targeting multiple proteins both through on-target and off-target binding. Network pharmacology models aim at addressing questions such as how and where in the disease network should one target to inhibit disease phenotypes, such as cancer growth, ideally leading to therapies that are less vulnerable to drug resistance and side effects by means of attacking the disease network at the systems level through synergistic and synthetic lethal interactions. Since the exponentially increasing number of potential drug target combinations makes pure experimental approach quickly unfeasible, this review depicts a number of computational models and algorithms that can effectively reduce the search space for determining the most promising combinations for experimental evaluation. Such computational-experimental strategies are geared toward realizing the full potential of multi-target treatments in different disease phenotypes. Our specific focus is on system-level network approaches to polypharmacology designs in anticancer drug discovery, where we give representative examples of how network-centric modeling may offer systematic strategies toward better understanding and even predicting the phenotypic responses to multi-target therapies.

  15. Social Grouping for Multi-Target Tracking and Head Pose Estimation in Video.

    PubMed

    Qin, Zhen; Shelton, Christian R

    2016-10-01

    Many computer vision tasks are more difficult when tackled without contextual information. For example, in multi-camera tracking, pedestrians may look very different in different cameras with varying pose and lighting conditions. Similarly, head direction estimation in high-angle surveillance video in which human head images are low resolution is challenging. Even humans can have trouble without contextual information. In this work, we couple novel contextual information, social grouping, with two important computer vision tasks: multi-target tracking and head pose/direction estimation in surveillance video. These three components are modeled in a probabilistic formulation and we provide effective solvers.We show that social grouping effectively helps to mitigate visual ambiguities in multi-camera tracking and head pose estimation. We further notice that in single-camera multi-target tracking, social grouping provides a natural high-order association cue that avoids existing complex algorithms for high-order track association. In experiments, we demonstrate improvements with our model over models without social grouping context and several state-of-art approaches on a number of publicly available datasets on tracking, head pose estimation, and group discovery.

  16. Antibacterial Drug Leads: DNA and Enzyme Multi-Targeting

    PubMed Central

    Zhu, Wei; Wang, Yang; Li, Kai; Gao, Jian; Huang, Chun-Hsiang; Chen, Chun-Chi; Ko, Tzu-Ping; Zhang, Yonghui; Guo, Rey-Ting; Oldfield, Eric

    2015-01-01

    We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG)2, and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100–500 nM and we found good correlations (R2 = 0.89, S. aureus; R2 = 0.79, E. coli)) between experimental and predicted cell growth inhibition by using DNA ΔTm and UPPS IC50 experimental results together with 1 computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multi-targeting. PMID:25574764

  17. Deterministic optimal maneuver strategy for multi-target missions

    NASA Technical Reports Server (NTRS)

    Dwivedi, N. P.

    1975-01-01

    This paper presents an optimal strategy for making impulsive correction to a multi-target trajectory by a single maneuver. The concept of an optimal maneuver time is introduced. The choice of suitable weighting functions is explored to enable one to properly translate the subjective desire of mission success into an objective cost function whose minimization yields the optimal strategy. It is shown that a number of strategies previously formulated are derivable from one general expression. A number of other interesting properties of the optimal strategy are described. Numerical results are presented for a typical two-target mission. It is shown that the strategy formulated is optimal. For some perturbations, there exists an optimal maneuver time different from the time of initiation of the perturbation. That is, the physical properties of the trajectory can be exploited to select the optimal time of making a corrective maneuver.

  18. RNA antisense purification (RAP) for mapping RNA interactions with chromatin.

    PubMed

    Engreitz, Jesse; Lander, Eric S; Guttman, Mitchell

    2015-01-01

    RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing.

  19. Multi-Robot, Multi-Target Particle Swarm Optimization Search in Noisy Wireless Environments

    SciTech Connect

    Kurt Derr; Milos Manic

    2009-05-01

    Multiple small robots (swarms) can work together using Particle Swarm Optimization (PSO) to perform tasks that are difficult or impossible for a single robot to accomplish. The problem considered in this paper is exploration of an unknown environment with the goal of finding a target(s) at an unknown location(s) using multiple small mobile robots. This work demonstrates the use of a distributed PSO algorithm with a novel adaptive RSS weighting factor to guide robots for locating target(s) in high risk environments. The approach was developed and analyzed on multiple robot single and multiple target search. The approach was further enhanced by the multi-robot-multi-target search in noisy environments. The experimental results demonstrated how the availability of radio frequency signal can significantly affect robot search time to reach a target.

  20. Multi-target pursuit formation of multi-agent systems

    NASA Astrophysics Data System (ADS)

    Yan, Jing; Guan, Xin-Ping; Luo, Xiao-Yuan

    2011-01-01

    The main goal of this paper is to design a team of agents that can accomplish multi-target pursuit formation using a developed leader—follower strategy. It is supposed that every target can accept a certain number of agents. First, each agent can automatically choose its target based on the distance from the agent to the target and the number of agents accepted by the target. In view of the fact that all agents are randomly dispersed in the workplace at the initial time, we present a numbering strategy for them. During the movement of agents, not every agent can always obtain pertinent state information about the targets. So, a developed leader—follower strategy and a pursuit formation algorithm are proposed. Under the proposed method, agents with the same target can maintain a circle formation. Furthermore, it turns out that the pursuit formation algorithm for agents to the desired formation is convergent. Simulation studies are provided to illustrate the effectiveness of the proposed method.

  1. A Network-Based Multi-Target Computational Estimation Scheme for Anticoagulant Activities of Compounds

    PubMed Central

    Li, Canghai; Chen, Lirong; Song, Jun; Tang, Yalin; Xu, Xiaojie

    2011-01-01

    Background Traditional virtual screening method pays more attention on predicted binding affinity between drug molecule and target related to a certain disease instead of phenotypic data of drug molecule against disease system, as is often less effective on discovery of the drug which is used to treat many types of complex diseases. Virtual screening against a complex disease by general network estimation has become feasible with the development of network biology and system biology. More effective methods of computational estimation for the whole efficacy of a compound in a complex disease system are needed, given the distinct weightiness of the different target in a biological process and the standpoint that partial inhibition of several targets can be more efficient than the complete inhibition of a single target. Methodology We developed a novel approach by integrating the affinity predictions from multi-target docking studies with biological network efficiency analysis to estimate the anticoagulant activities of compounds. From results of network efficiency calculation for human clotting cascade, factor Xa and thrombin were identified as the two most fragile enzymes, while the catalytic reaction mediated by complex IXa:VIIIa and the formation of the complex VIIIa:IXa were recognized as the two most fragile biological matter in the human clotting cascade system. Furthermore, the method which combined network efficiency with molecular docking scores was applied to estimate the anticoagulant activities of a serial of argatroban intermediates and eight natural products respectively. The better correlation (r = 0.671) between the experimental data and the decrease of the network deficiency suggests that the approach could be a promising computational systems biology tool to aid identification of anticoagulant activities of compounds in drug discovery. Conclusions This article proposes a network-based multi-target computational estimation method for

  2. IGF-1 Antisense Strategies for Cancer Treatment.

    PubMed

    Pan, Y X; Anthony, D D

    2000-01-01

    The technical approaches to gene therapy for cancer utilize ex vivo and in vivo gene-transfer methodology. This chapter focuses on applicability and use of an ex vivo approach using an IGF-1 antisense RNA strategy of treatment. Insulin-like growth factor 1 (IGF-1) and IGF-2 have pivotal roles in cell proliferation and development (for review, see 1-6). The preponderance of peptide synthesis and activity occur during fetal development, and protein synthesis is downregulated in most mature tissues except for adult liver. Further modulating the activities of these proteins are the levels of their respective cell-surface receptors and ligand-receptor interactions (3,5,6).

  3. Phenolic thio- and selenosemicarbazones as multi-target drugs.

    PubMed

    Calcatierra, Verónica; López, Óscar; Fernández-Bolaños, José G; Plata, Gabriela B; Padrón, José M

    2015-04-13

    A series of isosteric phenolic thio- and selenosemicarbazones have been obtained by condensation of naturally-occurring phenolic aldehydes and thio(seleno)semicarbazides. Title compounds were designed as potential multi-target drugs, and a series of structure-activity relationships could be established upon their in vitro assays: antioxidant activity, α-glucosidase inhibition and antiproliferative activity against six human tumor cell lines: A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (colon). For the antiradical activity, selenium atom and 2 or 3 phenolic hydroxyl groups proved to be essential motifs; remarkably, the compound with the most potent activity, with a trihydroxyphenyl scaffold (EC50 = 4.87 ± 1.57 μM) was found to be stronger than natural hydroxytyrosol, a potent antioxidant present in olive oil (EC50 = 13.80 ± 1.41 μM). Furthermore, one of the thiosemicarbazones was found to be a strong non-competitive inhibitor of α-glucosidase (Ki = 9.6 ± 1.6 μM), with an 8-fold increase in activity compared to acarbose (Ki = 77.9 ± 11.4 μM), marketed for the treatment of type-2 diabetes. Most of the synthesized compounds also exhibited relevant antiproliferative activities; in particular, seleno derivatives showed GI50 values lower than 6.0 μM for all the tested cell lines; N-naphthyl mono- and dihydroxylated derivatives behaved as more potent antiproliferative agents than 5-fluorouracil or cisplatin. PMID:25752525

  4. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    PubMed

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  5. The Antisense Transcriptome and the Human Brain.

    PubMed

    Mills, James D; Chen, Bei Jun; Ueberham, Uwe; Arendt, Thomas; Janitz, Michael

    2016-01-01

    The transcriptome of a cell is made up of a varied array of RNA species, including protein-coding RNAs, long non-coding RNAs, short non-coding RNAs, and circular RNAs. The cellular transcriptome is dynamic and can change depending on environmental factors, disease state and cellular context. The human brain has perhaps the most diverse transcriptome profile that is enriched for many species of RNA, including antisense transcripts. Antisense transcripts are produced when both the plus and minus strand of the DNA helix are transcribed at a particular locus. This results in an RNA transcript that has a partial or complete overlap with an intronic or exonic region of the sense transcript. While antisense transcription is known to occur at some level in most organisms, this review focuses specifically on antisense transcription in the brain and how regulation of genes by antisense transcripts can contribute to functional aspects of the healthy and diseased brain. First, we discuss different techniques that can be used in the identification and quantification of antisense transcripts. This is followed by examples of antisense transcription and modes of regulatory function that have been identified in the brain.

  6. Powerful inner/outer controlled multi-target magnetic nanoparticle drug carrier prepared by liquid photo-immobilization

    NASA Astrophysics Data System (ADS)

    Guan, Yan-Qing; Zheng, Zhe; Huang, Zheng; Li, Zhibin; Niu, Shuiqin; Liu, Jun-Ming

    2014-05-01

    Nanomagnetic materials offer exciting avenues for advancing cancer therapies. Most researches have focused on efficient delivery of drugs in the body by incorporating various drug molecules onto the surface of nanomagnetic particles. The challenge is how to synthesize low toxic nanocarriers with multi-target drug loading. The cancer cell death mechanisms associated with those nanocarriers remain unclear either. Following the cell biology mechanisms, we develop a liquid photo-immobilization approach to attach doxorubicin, folic acid, tumor necrosis factor-α, and interferon-γ onto the oleic acid molecules coated Fe3O4 magnetic nanoparticles to prepare a kind of novel inner/outer controlled multi-target magnetic nanoparticle drug carrier. In this work, this approach is demonstrated by a variety of structural and biomedical characterizations, addressing the anti-cancer effects in vivo and in vitro on the HeLa, and it is highly efficient and powerful in treating cancer cells in a valuable programmed cell death mechanism for overcoming drug resistance.

  7. Powerful inner/outer controlled multi-target magnetic nanoparticle drug carrier prepared by liquid photo-immobilization

    PubMed Central

    Guan, Yan-Qing; Zheng, Zhe; Huang, Zheng; Li, Zhibin; Niu, Shuiqin; Liu, Jun-Ming

    2014-01-01

    Nanomagnetic materials offer exciting avenues for advancing cancer therapies. Most researches have focused on efficient delivery of drugs in the body by incorporating various drug molecules onto the surface of nanomagnetic particles. The challenge is how to synthesize low toxic nanocarriers with multi-target drug loading. The cancer cell death mechanisms associated with those nanocarriers remain unclear either. Following the cell biology mechanisms, we develop a liquid photo-immobilization approach to attach doxorubicin, folic acid, tumor necrosis factor-α, and interferon-γ onto the oleic acid molecules coated Fe3O4 magnetic nanoparticles to prepare a kind of novel inner/outer controlled multi-target magnetic nanoparticle drug carrier. In this work, this approach is demonstrated by a variety of structural and biomedical characterizations, addressing the anti-cancer effects in vivo and in vitro on the HeLa, and it is highly efficient and powerful in treating cancer cells in a valuable programmed cell death mechanism for overcoming drug resistance. PMID:24845203

  8. Extending multi-tenant architectures: a database model for a multi-target support in SaaS applications

    NASA Astrophysics Data System (ADS)

    Rico, Antonio; Noguera, Manuel; Garrido, José Luis; Benghazi, Kawtar; Barjis, Joseph

    2016-05-01

    Multi-tenant architectures (MTAs) are considered a cornerstone in the success of Software as a Service as a new application distribution formula. Multi-tenancy allows multiple customers (i.e. tenants) to be consolidated into the same operational system. This way, tenants run and share the same application instance as well as costs, which are significantly reduced. Functional needs vary from one tenant to another; either companies from different sectors run different types of applications or, although deploying the same functionality, they do differ in the extent of their complexity. In any case, MTA leaves one major concern regarding the companies' data, their privacy and security, which requires special attention to the data layer. In this article, we propose an extended data model that enhances traditional MTAs in respect of this concern. This extension - called multi-target - allows MT applications to host, manage and serve multiple functionalities within the same multi-tenant (MT) environment. The practical deployment of this approach will allow SaaS vendors to target multiple markets or address different levels of functional complexity and yet commercialise just one single MT application. The applicability of the approach is demonstrated via a case study of a real multi-tenancy multi-target (MT2) implementation, called Globalgest.

  9. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  10. Curcumin: A multi-target disease-modifying agent for late-stage transthyretin amyloidosis

    PubMed Central

    Ferreira, Nelson; Gonçalves, Nádia P.; Saraiva, Maria J.; Almeida, Maria R.

    2016-01-01

    Transthyretin amyloidoses encompass a variety of acquired and hereditary diseases triggered by systemic extracellular accumulation of toxic transthyretin aggregates and fibrils, particularly in the peripheral nervous system. Since transthyretin amyloidoses are typically complex progressive disorders, therapeutic approaches aiming multiple molecular targets simultaneously, might improve therapy efficacy and treatment outcome. In this study, we evaluate the protective effect of physiologically achievable doses of curcumin on the cytotoxicity induced by transthyretin oligomers in vitro by showing reduction of caspase-3 activity and the levels of endoplasmic reticulum-resident chaperone binding immunoglobulin protein. When given to an aged Familial Amyloidotic Polyneuropathy mouse model, curcumin not only reduced transthyretin aggregates deposition and toxicity in both gastrointestinal tract and dorsal root ganglia but also remodeled congophilic amyloid material in tissues. In addition, curcumin enhanced internalization, intracellular transport and degradation of transthyretin oligomers by primary macrophages from aged Familial Amyloidotic Polyneuropathy transgenic mice, suggesting an impaired activation of naïve phagocytic cells exposed to transthyretin toxic intermediate species. Overall, our results clearly support curcumin or optimized derivatives as promising multi-target disease-modifying agent for late-stage transthyretin amyloidosis. PMID:27197872

  11. Multi-target QSAR and docking study of steroids binding to corticosteroid-binding globulin and sex hormone-binding globulin.

    PubMed

    Nikolic, Katarina; Filipic, Slavica; Agbaba, Danica

    2012-12-01

    The QSAR and docking studies were performed on fifty seven steroids with binding affinities for corticosteroid-binding globulin (CBG) and eighty four steroids with binding affinities for sex hormone-binding globulin (SHBG). Since the steroidal compounds have binding affinity for both CBG and SHBG, multi-target QSAR approach was employed to establish a unique QSAR method for simultaneous evaluation of the CBG and SHBG binding affinities. The constitutional, geometrical, physico-chemical and electronic descriptors were computed for the examined structures by use of the Chem3D Ultra 7.0.0, the Dragon 6.0, the MOPAC2009, and the Chemical Descriptors Library (CDL) program. Partial least squares regression (PLSR) has been applied for selection of the most relevant molecular descriptors and QSAR models building. The QSAR (SHGB) model, QSAR model (CBG), and multi-target QSAR model (CBG, SHBG) were created. The multi-target QSAR model (CBG and SHBG) was found to be more effective in describing the CBG and SHBG affinity of steroids in comparison to the one target models (QSAR (SHGB) model, QSAR model (CBG)). The multi-target QSAR study indicated the importance of the electronic descriptor (Mor16v), steric/symmetry descriptors (Eig06_EA(ed)), 2D autocorrelation descriptor (GATS4m), distance distribution descriptor (RDF045m), and atom type fingerprint descriptor (CDL-ATFP 253) in describing the CBG and SHBG affinity of steroidal compounds. Results of the created multi-target QSAR model were in accordance with the performed docking studies. The theoretical study defined physicochemical, electronic and structural requirements for selective and effective binding of steroids to the CBG and SHBG active sites.

  12. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic.

  13. Development of Antisense Drugs for Dyslipidemia.

    PubMed

    Yamamoto, Tsuyoshi; Wada, Fumito; Harada-Shiba, Mariko

    2016-09-01

    Abnormal elevation of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins in plasma as well as dysfunction of anti-atherogenic high-density lipoprotein (HDL) have both been recognized as essential components of the pathogenesis of atherosclerosis and are classified as dyslipidemia. This review describes the arc of development of antisense oligonucleotides for the treatment of dyslipidemia. Chemically-armed antisense candidates can act on various kinds of transcripts, including mRNA and miRNA, via several different endogenous antisense mechanisms, and have exhibited potent systemic anti-dyslipidemic effects. Here, we present specific cutting-edge technologies have recently been brought into antisense strategies, and describe how they have improved the potency of antisense drugs in regard to pharmacokinetics and pharmacodynamics. In addition, we discuss perspectives for the use of armed antisense oligonucleotides as new clinical options for dyslipidemia, in the light of outcomes of recent clinical trials and safety concerns indicated by several clinical and preclinical studies. PMID:27466159

  14. Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis

    PubMed Central

    2013-01-01

    Background The objective of this study was to report the use of multi-targeted polymerase chain reaction (PCR) in the diagnosis of presumed tubercular uveitis. Multi-targeted PCR using three targets specific for Mycobacterium tuberculosis, i.e., IS6110, MPB64, and protein b, was performed on intraocular fluid samples of 25 subjects. Nine had presumed tubercular uveitis, six had intraocular inflammation secondary to a nontubercular etiology (disease controls), and ten had no evidence of intraocular inflammation (normal controls). As described previously, response to antitubercular therapy was considered as the gold standard. Results Multi-targeted PCR was positive in seven out of nine patients with presumed tubercular uveitis and negative in all normal and disease controls. The sensitivity and specificity were 77.77% and 100%, respectively. For the diagnosis of presumed tubercular uveitis, multi-targeted PCR had a positive predictive value of 100% and a negative predictive value of 88.88%. Conclusion Multi-targeted PCR can be a valuable tool for diagnosing presumed tubercular uveitis. PMID:23514226

  15. Multi-Target Tracking Based on Multi-Bernoulli Filter with Amplitude for Unknown Clutter Rate.

    PubMed

    Yuan, Changshun; Wang, Jun; Lei, Peng; Bi, Yanxian; Sun, Zhongsheng

    2015-01-01

    Knowledge of the clutter rate is of critical importance in multi-target Bayesian tracking. However, estimating the clutter rate is a difficult problem in practice. In this paper, an improved multi-Bernoulli filter based on random finite sets for multi-target Bayesian tracking accommodating non-linear dynamic and measurement models, as well as unknown clutter rate, is proposed for radar sensors. The proposed filter incorporates the amplitude information into the state and measurement spaces to improve discrimination between actual targets and clutters, while adaptively generating the new-born object random finite sets using the measurements to eliminate reliance on prior random finite sets. A sequential Monte-Carlo implementation of the proposed filter is presented, and simulations are used to demonstrate the proposed filter's improvements in estimation accuracy of the target number and corresponding multi-target states, as well as the clutter rate. PMID:26690148

  16. Multi-Target Tracking Based on Multi-Bernoulli Filter with Amplitude for Unknown Clutter Rate.

    PubMed

    Yuan, Changshun; Wang, Jun; Lei, Peng; Bi, Yanxian; Sun, Zhongsheng

    2015-12-04

    Knowledge of the clutter rate is of critical importance in multi-target Bayesian tracking. However, estimating the clutter rate is a difficult problem in practice. In this paper, an improved multi-Bernoulli filter based on random finite sets for multi-target Bayesian tracking accommodating non-linear dynamic and measurement models, as well as unknown clutter rate, is proposed for radar sensors. The proposed filter incorporates the amplitude information into the state and measurement spaces to improve discrimination between actual targets and clutters, while adaptively generating the new-born object random finite sets using the measurements to eliminate reliance on prior random finite sets. A sequential Monte-Carlo implementation of the proposed filter is presented, and simulations are used to demonstrate the proposed filter's improvements in estimation accuracy of the target number and corresponding multi-target states, as well as the clutter rate.

  17. Multi-Target Tracking Based on Multi-Bernoulli Filter with Amplitude for Unknown Clutter Rate

    PubMed Central

    Yuan, Changshun; Wang, Jun; Lei, Peng; Bi, Yanxian; Sun, Zhongsheng

    2015-01-01

    Knowledge of the clutter rate is of critical importance in multi-target Bayesian tracking. However, estimating the clutter rate is a difficult problem in practice. In this paper, an improved multi-Bernoulli filter based on random finite sets for multi-target Bayesian tracking accommodating non-linear dynamic and measurement models, as well as unknown clutter rate, is proposed for radar sensors. The proposed filter incorporates the amplitude information into the state and measurement spaces to improve discrimination between actual targets and clutters, while adaptively generating the new-born object random finite sets using the measurements to eliminate reliance on prior random finite sets. A sequential Monte-Carlo implementation of the proposed filter is presented, and simulations are used to demonstrate the proposed filter’s improvements in estimation accuracy of the target number and corresponding multi-target states, as well as the clutter rate. PMID:26690148

  18. A modified high-intensity Cs sputter negative-ion source with multi-target mechanism

    NASA Astrophysics Data System (ADS)

    Houzhi, Si; Weizhong, Zhang; Jinhau, Zhu; Guangtian, Du; Tiaorong, Zhang; Xiang, Gao

    1993-04-01

    The source is based on Middleton's high-intensity mode, but modified to a multi-target version. It is equipped with a spherical molybdenum ionizer, a 20-position target wheel and a vacuum lock for loading and unloading sample batches. A metal-ceramic bonded section protected by a specially designed labyrinth shielding system results in reliable insulation of the cathode and convenient control of cesium vapor. The latter is particularly important when an oversupply of cesium occurs. The source was developed for accelerator mass spectrometry (AMS) applications. Recently, three versions based on the prototype of the source have been successfully tested to meet different requirements: (a) single target version, (b) multi-target version with manual sample change, and (c) multi-target version with remote control sample change. Some details of the technical and operational characteristics are presented.

  19. Cooperative motion control for multi-target observation

    SciTech Connect

    Parker, L.E.

    1997-08-01

    An important issue that arises in the automation of many security, surveillance, and reconnaissance tasks is that of monitoring (or observing) the movements of targets navigating in a bounded area of interest. A key research issue in these problems is that of sensor placement--determining where sensors should be located to maintain the targets in view. In complex applications involving limited-range sensors, the use of multiple sensors dynamically moving over time is required. In this paper, the author investigates the use of a cooperative team of autonomous sensor-based robots for the observation of multiple moving targets. The focus is primarily on developing the distributed control strategies that allow the robot team to attempt to minimize the total time in which targets escape observation by some robot team member in the area of interest. This paper first formalizes the problem and discusses related work. The author then presents a distributed approximate approach to solving this problem that combines low-level multi-robot control with higher-level reasoning control based on the ALLIANCE formalism. The effectiveness of the approach is analyzed by comparing it to three other feasible algorithms for cooperative control, showing the superiority of the approach for a large class of problems.

  20. Chemical Modification of the Multi-Target Neuroprotective Compound Fisetin

    PubMed Central

    Chiruta, Chandramouli; Schubert, David; Dargusch, Richard; Maher, Pamela

    2012-01-01

    Many factors are implicated in age-related CNS disorders making it unlikely that modulating only a single factor will provide effective treatment. Perhaps a better approach is to identify small molecules that have multiple biological activities relevant to the maintenance of brain function. Recently, we identified an orally active, neuroprotective and cognition-enhancing molecule, the flavonoid fisetin, that is effective in several animal models of CNS disorders. Fisetin has direct antioxidant activity and can also increase the intracellular levels of glutathione (GSH), the major endogenous antioxidant. In addition, fisetin has both neurotrophic and anti-inflammatory activity. However, its relatively high EC50 in cell based assays, low lipophilicity, high tPSA and poor bioavailability suggest that there is room for medicinal chemical improvement. Here we describe a multi-tiered approach to screening that has allowed us to identify fisetin derivatives with significantly enhanced activity in an in vitro neuroprotection model while at the same time maintaining other key activities. PMID:22192055

  1. Antisense therapeutics in oncology: current status

    PubMed Central

    Farooqi, Ammad Ahmad; Rehman, Zia ur; Muntane, Jordi

    2014-01-01

    There is increasing progress in translational oncology and tremendous breakthroughs have been made as evidenced by preclinical and clinical trials. Data obtained from high-throughput technologies are deepening our understanding about the molecular and gene network in cancer cells and rapidly emerging in vitro and in vivo evidence is highlighting the role of antisense agents as specific inhibitors of the expression of target genes, thus modulating the response of cancer cells to different therapeutic strategies. Much information is continuously being added into various facets of molecular oncology and it is now understood that overexpression of antiapoptotic proteins, oncogenes, oncogenic microRNAs (miRNA), and fusion proteins make cancer cells difficult to target. Delivery of antisense oligonucleotides has remained a challenge and technological developments have helped in overcoming hurdles by improving the ability to penetrate cells, effective and targeted binding to gene sequences, and downregulation of target gene function. Different delivery systems, including stable nucleic acid lipid particles, have shown potential in enhancing the delivery of cargo to the target site. In this review, we attempt to summarize the current progress in the development of antisense therapeutics and their potential in medical research. We partition this multicomponent review into introductory aspects about recent breakthroughs in antisense therapeutics. We also discuss how antisense therapeutics have shown potential in resensitizing resistant cancer cells to apoptosis by targeted inhibition of antiapoptotic proteins, oncogenic miRNAs, and BCR-ABL. PMID:25395862

  2. Distributed Multi-Target Tracking and Data Association in Vision Networks.

    PubMed

    Kamal, Ahmed T; Bappy, Jawadul H; Farrell, Jay A; Roy-Chowdhury, Amit K

    2016-07-01

    Distributed algorithms have recently gained immense popularity. With regards to computer vision applications, distributed multi-target tracking in a camera network is a fundamental problem. The goal is for all cameras to have accurate state estimates for all targets. Distributed estimation algorithms work by exchanging information between sensors that are communication neighbors. Vision-based distributed multi-target state estimation has at least two characteristics that distinguishes it from other applications. First, cameras are directional sensors and often neighboring sensors may not be sensing the same targets, i.e., they are naive with respect to that target. Second, in the presence of clutter and multiple targets, each camera must solve a data association problem. This paper presents an information-weighted, consensus-based, distributed multi-target tracking algorithm referred to as the Multi-target Information Consensus (MTIC) algorithm that is designed to address both the naivety and the data association problems. It converges to the centralized minimum mean square error estimate. The proposed MTIC algorithm and its extensions to non-linear camera models, termed as the Extended MTIC (EMTIC), are robust to false measurements and limited resources like power, bandwidth and the real-time operational requirements. Simulation and experimental analysis are provided to support the theoretical results.

  3. Distributed Multi-Target Tracking and Data Association in Vision Networks.

    PubMed

    Kamal, Ahmed T; Bappy, Jawadul H; Farrell, Jay A; Roy-Chowdhury, Amit K

    2016-07-01

    Distributed algorithms have recently gained immense popularity. With regards to computer vision applications, distributed multi-target tracking in a camera network is a fundamental problem. The goal is for all cameras to have accurate state estimates for all targets. Distributed estimation algorithms work by exchanging information between sensors that are communication neighbors. Vision-based distributed multi-target state estimation has at least two characteristics that distinguishes it from other applications. First, cameras are directional sensors and often neighboring sensors may not be sensing the same targets, i.e., they are naive with respect to that target. Second, in the presence of clutter and multiple targets, each camera must solve a data association problem. This paper presents an information-weighted, consensus-based, distributed multi-target tracking algorithm referred to as the Multi-target Information Consensus (MTIC) algorithm that is designed to address both the naivety and the data association problems. It converges to the centralized minimum mean square error estimate. The proposed MTIC algorithm and its extensions to non-linear camera models, termed as the Extended MTIC (EMTIC), are robust to false measurements and limited resources like power, bandwidth and the real-time operational requirements. Simulation and experimental analysis are provided to support the theoretical results. PMID:26441444

  4. Antisense-mediated exon skipping: taking advantage of a trick from Mother Nature to treat rare genetic diseases.

    PubMed

    Veltrop, Marcel; Aartsma-Rus, Annemieke

    2014-07-01

    Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense oligonucleotide treatment to the splicing thus has therapeutic potential for many rare diseases. In this review we will focus on the state of the art on exon skipping using antisense oligonucleotides as a potential therapy for rare genetic diseases, outlining how this versatile approach can be exploited to correct for different mutations.

  5. Functionalization of an Antisense Small RNA

    PubMed Central

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  6. Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration

    PubMed Central

    Ramsay, Rona R.; Majekova, Magdalena; Medina, Milagros; Valoti, Massimo

    2016-01-01

    HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to “dirty drugs” for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are

  7. Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration

    PubMed Central

    Ramsay, Rona R.; Majekova, Magdalena; Medina, Milagros; Valoti, Massimo

    2016-01-01

    HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to “dirty drugs” for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are

  8. Key Targets for Multi-Target Ligands Designed to Combat Neurodegeneration.

    PubMed

    Ramsay, Rona R; Majekova, Magdalena; Medina, Milagros; Valoti, Massimo

    2016-01-01

    HIGHLIGHTS Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration.Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL).ASS234 is an irreversible inhibitor of MAO A >MAO B and has micromolar potency against the cholinesterases.ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite.SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium.Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to "dirty drugs" for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson's Disease's (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are simply

  9. Development of antisense oligodeoxynucleotides for transplantation.

    PubMed

    Stepkowski, S M

    2000-06-01

    Over last ten years antisense technology has been improved to provide powerful tools to selectively inhibit production of different mRNAs. This technology has been applied in transplantation to prolong the survival of organ allografts and to prevent development of ischemic/reperfusion injury in grafts. The present review describes technological progress in chemical modifications from antisense phosphodiester oligonucleotides to phosphorothioate oligonucleotides and the most advanced chimeric oligonucleotides with methoxyethyl groups attached at both ends or at one end of the oligonucleotide. Results indicate that phosphorothioate oligonucleotides, designed to block intercellular adhesion molecule-1 (ICAM-1), extended the survival of heart and kidney allografts when administered to donors or recipients. Combination of ICAM-1 antisense phosphorothioate oligonucleotide and cyclosporine (CsA) produced a potent synergistic interaction on allograft survival in comparison with each drug alone. The same ICAM-1 phosphorothioate oligonucleotide used for perfusion of kidney grafts prevented development of ischemic/reperfusion injury. We also compared the effect of c-raf mRNA inhibition on heart allograft survival by phosphorothioate oligonucleotide or phosphorothioate/methoxyethyl oligonucleotide used alone or in combination with CsA or sirolimus (SRL). The results documented that addition of methoxyethyl modifications at both ends or at one end of oligonucleotides significantly improved the in vivo antisense activity. Combined therapy with c-raf antisense phosphorothioate/methoxyethyl oligonucleotide and SRL synergistically extended the survival of heart allografts. Thus, antisense technology may provide not only tools to examine the effects of selective inhibition of different molecules involved in allograft rejection but also act as potential therapeutic agents.

  10. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  11. Strand-specific community RNA-seq reveals prevalent and dynamic antisense transcription in human gut microbiota

    PubMed Central

    Bao, Guanhui; Wang, Mingjie; Doak, Thomas G.; Ye, Yuzhen

    2015-01-01

    Metagenomics and other meta-omics approaches (including metatranscriptomics) provide insights into the composition and function of microbial communities living in different environments or animal hosts. Metatranscriptomics research provides an unprecedented opportunity to examine gene regulation for many microbial species simultaneously, and more importantly, for the majority that are unculturable microbial species, in their natural environments (or hosts). Current analyses of metatranscriptomic datasets focus on the detection of gene expression levels and the study of the relationship between changes of gene expression and changes of environment. As a demonstration of utilizing metatranscriptomics beyond these common analyses, we developed a computational and statistical procedure to analyze the antisense transcripts in strand-specific metatranscriptomic datasets. Antisense RNAs encoded on the DNA strand opposite a gene’s CDS have the potential to form extensive base-pairing interactions with the corresponding sense RNA, and can have important regulatory functions. Most studies of antisense RNAs in bacteria are rather recent, are mostly based on transcriptome analysis, and have been applied mainly to single bacterial species. Application of our approaches to human gut-associated metatranscriptomic datasets allowed us to survey antisense transcription for a large number of bacterial species associated with human beings. The ratio of protein coding genes with antisense transcription ranges from 0 to 35.8% (median = 10.0%) among 47 species. Our results show that antisense transcription is dynamic, varying between human individuals. Functional enrichment analysis revealed a preference of certain gene functions for antisense transcription, and transposase genes are among the most prominent ones (but we also observed antisense transcription in bacterial house-keeping genes). PMID:26388849

  12. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  13. Chemical modification study of antisense gapmers.

    PubMed

    Stanton, Robert; Sciabola, Simone; Salatto, Christopher; Weng, Yan; Moshinsky, Debra; Little, Jeremy; Walters, Evan; Kreeger, John; DiMattia, Debra; Chen, Tracy; Clark, Tracey; Liu, Mei; Qian, Jessie; Roy, Marc; Dullea, Robert

    2012-10-01

    A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic--using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.

  14. Antisense DNAs as multisite genomic modulators identified by DNA microarray

    PubMed Central

    Cho, Yee Sook; Kim, Meyoung-Kon; Cheadle, Chris; Neary, Catherine; Becker, Kevin G.; Cho-Chung, Yoon S.

    2001-01-01

    Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIα alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation. PMID:11481453

  15. Systematic mining of analog series with related core structures in multi-target activity space.

    PubMed

    Gupta-Ostermann, Disha; Hu, Ye; Bajorath, Jürgen

    2013-08-01

    We have aimed to systematically extract analog series with related core structures from multi-target activity space to explore target promiscuity of closely related analogous. Therefore, a previously introduced SAR matrix structure was adapted and further extended for large-scale data mining. These matrices organize analog series with related yet distinct core structures in a consistent manner. High-confidence compound activity data yielded more than 2,300 non-redundant matrices capturing 5,821 analog series that included 4,288 series with multi-target and 735 series with multi-family activities. Many matrices captured more than three analog series with activity against more than five targets. The matrices revealed a variety of promiscuity patterns. Compound series matrices also contain virtual compounds, which provide suggestions for compound design focusing on desired activity profiles.

  16. Cardinality Balanced Multi-Target Multi-Bernoulli Filter with Error Compensation

    PubMed Central

    He, Xiangyu; Liu, Guixi

    2016-01-01

    The cardinality balanced multi-target multi-Bernoulli (CBMeMBer) filter developed recently has been proved an effective multi-target tracking (MTT) algorithm based on the random finite set (RFS) theory, and it can jointly estimate the number of targets and their states from a sequence of sensor measurement sets. However, because of the existence of systematic errors in sensor measurements, the CBMeMBer filter can easily produce different levels of performance degradation. In this paper, an extended CBMeMBer filter, in which the joint probability density function of target state and systematic error is recursively estimated, is proposed to address the MTT problem based on the sensor measurements with systematic errors. In addition, an analytic implementation of the extended CBMeMBer filter is also presented for linear Gaussian models. Simulation results confirm that the proposed algorithm can track multiple targets with better performance. PMID:27589764

  17. A detection method for infrared multi-target in aerospace backgound

    NASA Astrophysics Data System (ADS)

    Wang, Ningming; Zhang, Yazhou

    2015-11-01

    Main task of the infrared search and track system is analyzing and identifying targets of airspace. But first this is needed to detect all targets in infrared image. Therefore, the multi-target detection algorithms are studied and we propose an effective multi-target detection method. Firstly, an improved morphological operator is designed based on airspace background and target traits of infrared image. Background is weakened but targets are enhanced when infrared image is processed by the gray morphological filter. Then, potential targets are found by the maximum local sum algorithm. Finally, true targets are affirmed based on data association of sequence images. The infrared images got from long-wavelength infrared camera are processed with the method of the paper. Experiment results show that the method can detect targets in infrared image quickly and accurately.

  18. Cardinality Balanced Multi-Target Multi-Bernoulli Filter with Error Compensation.

    PubMed

    He, Xiangyu; Liu, Guixi

    2016-08-31

    The cardinality balanced multi-target multi-Bernoulli (CBMeMBer) filter developed recently has been proved an effective multi-target tracking (MTT) algorithm based on the random finite set (RFS) theory, and it can jointly estimate the number of targets and their states from a sequence of sensor measurement sets. However, because of the existence of systematic errors in sensor measurements, the CBMeMBer filter can easily produce different levels of performance degradation. In this paper, an extended CBMeMBer filter, in which the joint probability density function of target state and systematic error is recursively estimated, is proposed to address the MTT problem based on the sensor measurements with systematic errors. In addition, an analytic implementation of the extended CBMeMBer filter is also presented for linear Gaussian models. Simulation results confirm that the proposed algorithm can track multiple targets with better performance.

  19. Cardinality Balanced Multi-Target Multi-Bernoulli Filter with Error Compensation.

    PubMed

    He, Xiangyu; Liu, Guixi

    2016-01-01

    The cardinality balanced multi-target multi-Bernoulli (CBMeMBer) filter developed recently has been proved an effective multi-target tracking (MTT) algorithm based on the random finite set (RFS) theory, and it can jointly estimate the number of targets and their states from a sequence of sensor measurement sets. However, because of the existence of systematic errors in sensor measurements, the CBMeMBer filter can easily produce different levels of performance degradation. In this paper, an extended CBMeMBer filter, in which the joint probability density function of target state and systematic error is recursively estimated, is proposed to address the MTT problem based on the sensor measurements with systematic errors. In addition, an analytic implementation of the extended CBMeMBer filter is also presented for linear Gaussian models. Simulation results confirm that the proposed algorithm can track multiple targets with better performance. PMID:27589764

  20. Microfluidic immunomagnetic multi-target sorting--a model for controlling deflection of paramagnetic beads.

    PubMed

    Tsai, Scott S H; Griffiths, Ian M; Stone, Howard A

    2011-08-01

    We describe a microfluidic system that uses a magnetic field to sort paramagnetic beads by deflecting them in the direction normal to the flow. In the experiments we systematically study the dependence of the beads' deflection on bead size and susceptibility, magnet strength, fluid speed and viscosity, and device geometry. We also develop a design parameter that can aid in the design of microfluidic devices for immunomagnetic multi-target sorting. PMID:21677937

  1. Dielectrophoresis-based classification of cells using multi-target multiple-hypothesis tracking.

    PubMed

    Dickerson, Samuel J; Chiarulli, Donald M; Levitan, Steven P; Carthel, Craig; Coraluppi, Stefano

    2014-01-01

    In this paper we present a novel methodology for classifying cells by using a combination of dielectrophoresis, image tracking and classification algorithms. We use dielectrophoresis to induce unique motion patterns in cells of interest. Motion is extracted via multi-target multiple-hypothesis tracking. Trajectories are then used to classify cells based on a generalized likelihood ratio test. We present results of a simulation study and of our prototype tracking the dielectrophoretic velocities of cells. PMID:25570230

  2. Dielectrophoresis-based classification of cells using multi-target multiple-hypothesis tracking.

    PubMed

    Dickerson, Samuel J; Chiarulli, Donald M; Levitan, Steven P; Carthel, Craig; Coraluppi, Stefano

    2014-01-01

    In this paper we present a novel methodology for classifying cells by using a combination of dielectrophoresis, image tracking and classification algorithms. We use dielectrophoresis to induce unique motion patterns in cells of interest. Motion is extracted via multi-target multiple-hypothesis tracking. Trajectories are then used to classify cells based on a generalized likelihood ratio test. We present results of a simulation study and of our prototype tracking the dielectrophoretic velocities of cells.

  3. Identification and characterization of carprofen as a multi-target FAAH/COX inhibitor

    PubMed Central

    Favia, Angelo D.; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2013-01-01

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the non-steroid anti-inflammatory drug, carprofen, as a multi-target-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2 and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several racemic derivatives of carprofen, sharing this multi-target activity. This may result in improved analgesic efficacy and reduced side effects (Naidu, et al (2009) J Pharmacol Exp Ther 329, 48-56; Fowler, C.J. et al. (2012) J Enzym Inhib Med Chem Jan 6; Sasso, et al (2012) Pharmacol Res 65, 553). The new compounds are among the most potent multi-target FAAH/COXs inhibitors reported so far in the literature, and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs. PMID:23043222

  4. Composition of photosystem II antenna in light-harvesting complex II antisense tobacco plants at varying irradiances.

    PubMed Central

    Flachmann, R

    1997-01-01

    Plants with genes coding for chlorophyll a/b-binding proteins of light-harvesting complex II (LHCII) in antisense orientation (Lhcb) that are characterized by severely reduced Lhcb transcript levels (below 10% of wild type) do not show a bleached phenotype due to a specific loss of the polypeptide. To produce such a phenotype, a conceptually different antisense approach was tested with a dual-functional transcript encoding the gene for hygromycin phosphotransferase and the transit sequence of Lhcb1-2 in the antisense orientation. Using increasing concentrations of hygromycin, transformants with Lhcb steady-state levels as low as 9% of wild type were regenerated and grown in a growth chamber. Together with Lhcb antisense plants obtained in an earlier study, these antisense plants were analyzed biochemically for their photosystem II (PSII) antenna composition under varying light conditions. All antisense plants showed a characteristic low-irradiance-induced increase of their PSII antenna size as determined by higher chlorophyll concentrations, an increased content of LHCII, and a constant chlorophyll b-to-lutein ratio in comparison with control plants. One to 5% of the total Lhcb transcript amount was sufficient to allow unrestricted formation of the PSII antenna at low irradiance, suggesting that LHCH biogenesis is not controlled primarily by transcription. PMID:9085572

  5. Network pharmacology of cancer: From understanding of complex interactomes to the design of multi-target specific therapeutics from nature.

    PubMed

    Poornima, Paramasivan; Kumar, Jothi Dinesh; Zhao, Qiaoli; Blunder, Martina; Efferth, Thomas

    2016-09-01

    Despite massive investments in drug research and development, the significant decline in the number of new drugs approved or translated to clinical use raises the question, whether single targeted drug discovery is the right approach. To combat complex systemic diseases that harbour robust biological networks such as cancer, single target intervention is proved to be ineffective. In such cases, network pharmacology approaches are highly useful, because they differ from conventional drug discovery by addressing the ability of drugs to target numerous proteins or networks involved in a disease. Pleiotropic natural products are one of the promising strategies due to their multi-targeting and due to lower side effects. In this review, we discuss the application of network pharmacology for cancer drug discovery. We provide an overview of the current state of knowledge on network pharmacology, focus on different technical approaches and implications for cancer therapy (e.g. polypharmacology and synthetic lethality), and illustrate the therapeutic potential with selected examples green tea polyphenolics, Eleutherococcus senticosus, Rhodiola rosea, and Schisandra chinensis). Finally, we present future perspectives on their plausible applications for diagnosis and therapy of cancer. PMID:27329331

  6. Chemosensitization by antisense oligonucleotides targeting MDM2.

    PubMed

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  7. Using both strands: The fundamental nature of antisense transcription.

    PubMed

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  8. Multi-target drugs: the trend of drug research and development.

    PubMed

    Lu, Jin-Jian; Pan, Wei; Hu, Yuan-Jia; Wang, Yi-Tao

    2012-01-01

    Summarizing the status of drugs in the market and examining the trend of drug research and development is important in drug discovery. In this study, we compared the drug targets and the market sales of the new molecular entities approved by the U.S. Food and Drug Administration from January 2000 to December 2009. Two networks, namely, the target-target and drug-drug networks, have been set up using the network analysis tools. The multi-target drugs have much more potential, as shown by the network visualization and the market trends. We discussed the possible reasons and proposed the rational strategies for drug research and development in the future.

  9. Multi-Target State Extraction for the SMC-PHD Filter

    PubMed Central

    Si, Weijian; Wang, Liwei; Qu, Zhiyu

    2016-01-01

    The sequential Monte Carlo probability hypothesis density (SMC-PHD) filter has been demonstrated to be a favorable method for multi-target tracking. However, the time-varying target states need to be extracted from the particle approximation of the posterior PHD, which is difficult to implement due to the unknown relations between the large amount of particles and the PHD peaks representing potential target locations. To address this problem, a novel multi-target state extraction algorithm is proposed in this paper. By exploiting the information of measurements and particle likelihoods in the filtering stage, we propose a validation mechanism which aims at selecting effective measurements and particles corresponding to detected targets. Subsequently, the state estimates of the detected and undetected targets are performed separately: the former are obtained from the particle clusters directed by effective measurements, while the latter are obtained from the particles corresponding to undetected targets via clustering method. Simulation results demonstrate that the proposed method yields better estimation accuracy and reliability compared to existing methods. PMID:27322274

  10. Pharmacological Characterization of Memoquin, a Multi-Target Compound for the Treatment of Alzheimer's Disease

    PubMed Central

    Capurro, Valeria; Busquet, Perrine; Lopes, Joao Pedro; Bertorelli, Rosalia; Tarozzo, Glauco; Bolognesi, Maria Laura; Piomelli, Daniele; Reggiani, Angelo; Cavalli, Andrea

    2013-01-01

    Alzheimer's disease (AD) is characterized by progressive loss of cognitive function, dementia and altered behavior. Over 30 million people worldwide suffer from AD and available therapies are still palliative rather than curative. Recently, Memoquin (MQ), a quinone-bearing polyamine compound, has emerged as a promising anti-AD lead candidate, mainly thanks to its multi-target profile. MQ acts as an acetylcholinesterase and β-secretase-1 inhibitor, and also possesses anti-amyloid and anti-oxidant properties. Despite this potential interest, in vivo behavioral studies with MQ have been limited. Here, we report on in vivo studies with MQ (acute and sub-chronic treatments; 7–15 mg/kg per os) carried out using two different mouse models: i) scopolamine- and ii) beta-amyloid peptide- (Aβ-) induced amnesia. Several aspects related to memory were examined using the T-maze, the Morris water maze, the novel object recognition, and the passive avoidance tasks. At the dose of 15 mg/kg, MQ was able to rescue all tested aspects of cognitive impairment including spatial, episodic, aversive, short and long-term memory in both scopolamine- and Aβ-induced amnesia models. Furthermore, when tested in primary cortical neurons, MQ was able to fully prevent the Aβ-induced neurotoxicity mediated by oxidative stress. The results support the effectiveness of MQ as a cognitive enhancer, and highlight the value of a multi-target strategy to address the complex nature of cognitive dysfunction in AD. PMID:23441223

  11. Multi-Target State Extraction for the SMC-PHD Filter.

    PubMed

    Si, Weijian; Wang, Liwei; Qu, Zhiyu

    2016-01-01

    The sequential Monte Carlo probability hypothesis density (SMC-PHD) filter has been demonstrated to be a favorable method for multi-target tracking. However, the time-varying target states need to be extracted from the particle approximation of the posterior PHD, which is difficult to implement due to the unknown relations between the large amount of particles and the PHD peaks representing potential target locations. To address this problem, a novel multi-target state extraction algorithm is proposed in this paper. By exploiting the information of measurements and particle likelihoods in the filtering stage, we propose a validation mechanism which aims at selecting effective measurements and particles corresponding to detected targets. Subsequently, the state estimates of the detected and undetected targets are performed separately: the former are obtained from the particle clusters directed by effective measurements, while the latter are obtained from the particles corresponding to undetected targets via clustering method. Simulation results demonstrate that the proposed method yields better estimation accuracy and reliability compared to existing methods. PMID:27322274

  12. Multi-Target State Extraction for the SMC-PHD Filter.

    PubMed

    Si, Weijian; Wang, Liwei; Qu, Zhiyu

    2016-06-17

    The sequential Monte Carlo probability hypothesis density (SMC-PHD) filter has been demonstrated to be a favorable method for multi-target tracking. However, the time-varying target states need to be extracted from the particle approximation of the posterior PHD, which is difficult to implement due to the unknown relations between the large amount of particles and the PHD peaks representing potential target locations. To address this problem, a novel multi-target state extraction algorithm is proposed in this paper. By exploiting the information of measurements and particle likelihoods in the filtering stage, we propose a validation mechanism which aims at selecting effective measurements and particles corresponding to detected targets. Subsequently, the state estimates of the detected and undetected targets are performed separately: the former are obtained from the particle clusters directed by effective measurements, while the latter are obtained from the particles corresponding to undetected targets via clustering method. Simulation results demonstrate that the proposed method yields better estimation accuracy and reliability compared to existing methods.

  13. Multi-target-qubit unconventional geometric phase gate in a multi-cavity system.

    PubMed

    Liu, Tong; Cao, Xiao-Zhi; Su, Qi-Ping; Xiong, Shao-Jie; Yang, Chui-Ping

    2016-01-01

    Cavity-based large scale quantum information processing (QIP) may involve multiple cavities and require performing various quantum logic operations on qubits distributed in different cavities. Geometric-phase-based quantum computing has drawn much attention recently, which offers advantages against inaccuracies and local fluctuations. In addition, multiqubit gates are particularly appealing and play important roles in QIP. We here present a simple and efficient scheme for realizing a multi-target-qubit unconventional geometric phase gate in a multi-cavity system. This multiqubit phase gate has a common control qubit but different target qubits distributed in different cavities, which can be achieved using a single-step operation. The gate operation time is independent of the number of qubits and only two levels for each qubit are needed. This multiqubit gate is generic, e.g., by performing single-qubit operations, it can be converted into two types of significant multi-target-qubit phase gates useful in QIP. The proposal is quite general, which can be used to accomplish the same task for a general type of qubits such as atoms, NV centers, quantum dots, and superconducting qubits. PMID:26898176

  14. Multi-Targeted Antithrombotic Therapy for Total Artificial Heart Device Patients

    PubMed Central

    Ramirez, Angeleah; Riley, Jeffrey B.; Joyce, Lyle D.

    2016-01-01

    Abstract: To prevent thrombotic or bleeding events in patients receiving a total artificial heart (TAH), agents have been used to avoid adverse events. The purpose of this article is to outline the adoption and results of a multi-targeted antithrombotic clinical procedure guideline (CPG) for TAH patients. Based on literature review of TAH anticoagulation and multiple case series, a CPG was designed to prescribe the use of multiple pharmacological agents. Total blood loss, Thromboelastograph® (TEG), and platelet light-transmission aggregometry (LTA) measurements were conducted on 13 TAH patients during the first 2 weeks of support in our institution. Target values and actual medians for postimplant days 1, 3, 7, and 14 were calculated for kaolinheparinase TEG, kaolin TEG, LTA, and estimated blood loss. Protocol guidelines were followed and anticoagulation management reduced bleeding and prevented thrombus formation as well as thromboembolic events in TAH patients postimplantation. The patients in this study were susceptible to a variety of possible complications such as mechanical device issues, thrombotic events, infection, and bleeding. Among them all it was clear that patients were at most risk for bleeding, particularly on postoperative days 1 through 3. However, bleeding was reduced into postoperative days 3 and 7, indicating that acceptable hemostasis was achieved with the anticoagulation protocol. The multidisciplinary, multi-targeted anticoagulation clinical procedure guideline was successful to maintain adequate antithrombotic therapy for TAH patients. PMID:27134306

  15. Multi-Targeted Antithrombotic Therapy for Total Artificial Heart Device Patients.

    PubMed

    Ramirez, Angeleah; Riley, Jeffrey B; Joyce, Lyle D

    2016-03-01

    To prevent thrombotic or bleeding events in patients receiving a total artificial heart (TAH), agents have been used to avoid adverse events. The purpose of this article is to outline the adoption and results of a multi-targeted antithrombotic clinical procedure guideline (CPG) for TAH patients. Based on literature review of TAH anticoagulation and multiple case series, a CPG was designed to prescribe the use of multiple pharmacological agents. Total blood loss, Thromboelastograph(®) (TEG), and platelet light-transmission aggregometry (LTA) measurements were conducted on 13 TAH patients during the first 2 weeks of support in our institution. Target values and actual medians for postimplant days 1, 3, 7, and 14 were calculated for kaolinheparinase TEG, kaolin TEG, LTA, and estimated blood loss. Protocol guidelines were followed and anticoagulation management reduced bleeding and prevented thrombus formation as well as thromboembolic events in TAH patients postimplantation. The patients in this study were susceptible to a variety of possible complications such as mechanical device issues, thrombotic events, infection, and bleeding. Among them all it was clear that patients were at most risk for bleeding, particularly on postoperative days 1 through 3. However, bleeding was reduced into postoperative days 3 and 7, indicating that acceptable hemostasis was achieved with the anticoagulation protocol. The multidisciplinary, multi-targeted anticoagulation clinical procedure guideline was successful to maintain adequate antithrombotic therapy for TAH patients.

  16. ASS234, As a New Multi-Target Directed Propargylamine for Alzheimer's Disease Therapy

    PubMed Central

    Marco-Contelles, José; Unzeta, Mercedes; Bolea, Irene; Esteban, Gerard; Ramsay, Rona R.; Romero, Alejandro; Martínez-Murillo, Ricard; Carreiras, M. Carmo; Ismaili, Lhassane

    2016-01-01

    Highlights: ASS2324 is a hybrid compound resulting from the juxtaposition of donepezil and the propargylamine PF9601NASS2324 is a multi-target directed propargylamine able to bind to all the AChE/BuChE and MAO A/B enzymesASS2324 shows antioxidant, neuroprotective and suitable permeability propertiesASS2324 restores the scopolamine-induced cognitive impairment to the same extent as donepezil, and is less toxicASS2324 prevents β-amyloid induced aggregation in the cortex of double transgenic miceASS2324 is the most advanced anti-Alzheimer agent for pre-clinical studies that we have identified in our laboratories The complex nature of Alzheimer's disease (AD) has prompted the design of Multi-Target-Directed Ligands (MTDL) able to bind to diverse biochemical targets involved in the progress and development of the disease. In this context, we have designed a number of MTD propargylamines (MTDP) showing antioxidant, anti-beta-amyloid, anti-inflammatory, as well as cholinesterase and monoamine oxidase (MAO) inhibition capacities. Here, we describe these properties in the MTDL ASS234, our lead-compound ready to enter in pre-clinical studies for AD, as a new multipotent, permeable cholinesterase/monoamine oxidase inhibitor, able to inhibit Aβ-aggregation, and possessing antioxidant and neuroprotective properties. PMID:27445665

  17. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  18. Antisense transcription as a tool to tune gene expression.

    PubMed

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  19. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    SciTech Connect

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  20. Inhibition of the alternative complement pathway by antisense oligonucleotides targeting complement factor B improves lupus nephritis in mice.

    PubMed

    Grossman, Tamar R; Hettrick, Lisa A; Johnson, Robert B; Hung, Gene; Peralta, Raechel; Watt, Andrew; Henry, Scott P; Adamson, Peter; Monia, Brett P; McCaleb, Michael L

    2016-06-01

    Systemic lupus erythematosus is an autoimmune disease that manifests in widespread complement activation and deposition of complement fragments in the kidney. The complement pathway is believed to play a significant role in the pathogenesis and in the development of lupus nephritis. Complement factor B is an important activator of the alternative complement pathway and increasing evidence supports reducing factor B as a potential novel therapy to lupus nephritis. Here we investigated whether pharmacological reduction of factor B expression using antisense oligonucleotides could be an effective approach for the treatment of lupus nephritis. We identified potent and well tolerated factor B antisense oligonucleotides that resulted in significant reductions in hepatic and plasma factor B levels when administered to normal mice. To test the effects of factor B antisense oligonucleotides on lupus nephritis, we used two different mouse models, NZB/W F1 and MRL/lpr mice, that exhibit lupus nephritis like renal pathology. Antisense oligonucleotides mediated reductions in circulating factor B levels were associated with significant improvements in renal pathology, reduced glomerular C3 deposition and proteinuria, and improved survival. These data support the strategy of using factor B antisense oligonucleotides for treatment of lupus nephritis in humans.

  1. Representation of multi-target activity landscapes through target pair-based compound encoding in self-organizing maps.

    PubMed

    Iyer, Preeti; Bajorath, Jürgen

    2011-11-01

    Activity landscape representations provide access to structure-activity relationships information in compound data sets. In general, activity landscape models integrate molecular similarity relationships with biological activity data. Typically, activity against a single target is monitored. However, for steadily increasing numbers of compounds, activity against multiple targets is reported, resulting in an opportunity, and often a need, to explore multi-target structure-activity relationships. It would be attractive to utilize activity landscape representations to aid in this process, but the design of activity landscapes for multiple targets is a complicated task. Only recently has a first multi-target landscape model been introduced, consisting of an annotated compound network focused on the systematic detection of activity cliffs. Herein, we report a conceptually different multi-target activity landscape design that is based on a 2D projection of chemical reference space using self-organizing maps and encodes compounds as arrays of pair-wise target activity relationships. In this context, we introduce the concept of discontinuity in multi-target activity space. The well-ordered activity landscape model highlights centers of discontinuity in activity space and is straightforward to interpret. It has been applied to analyze compound data sets with three, four, and five target annotations and identify multi-target structure-activity relationships determinants in analog series.

  2. Improved Bearings-Only Multi-Target Tracking with GM-PHD Filtering.

    PubMed

    Zhang, Qian; Song, Taek Lyul

    2016-01-01

    In this paper, an improved nonlinear Gaussian mixture probability hypothesis density (GM-PHD) filter is proposed to address bearings-only measurements in multi-target tracking. The proposed method, called the Gaussian mixture measurements-probability hypothesis density (GMM-PHD) filter, not only approximates the posterior intensity using a Gaussian mixture, but also models the likelihood function with a Gaussian mixture instead of a single Gaussian distribution. Besides, the target birth model of the GMM-PHD filter is assumed to be partially uniform instead of a Gaussian mixture. Simulation results show that the proposed filter outperforms the GM-PHD filter embedded with the extended Kalman filter (EKF) and the unscented Kalman filter (UKF). PMID:27626423

  3. Improved Bearings-Only Multi-Target Tracking with GM-PHD Filtering.

    PubMed

    Zhang, Qian; Song, Taek Lyul

    2016-09-10

    In this paper, an improved nonlinear Gaussian mixture probability hypothesis density (GM-PHD) filter is proposed to address bearings-only measurements in multi-target tracking. The proposed method, called the Gaussian mixture measurements-probability hypothesis density (GMM-PHD) filter, not only approximates the posterior intensity using a Gaussian mixture, but also models the likelihood function with a Gaussian mixture instead of a single Gaussian distribution. Besides, the target birth model of the GMM-PHD filter is assumed to be partially uniform instead of a Gaussian mixture. Simulation results show that the proposed filter outperforms the GM-PHD filter embedded with the extended Kalman filter (EKF) and the unscented Kalman filter (UKF).

  4. [Possibilities for inhibiting tumor-induced angiogenesis: results with multi-target tyrosine kinase inhibitors].

    PubMed

    Török, Szilvia; Döme, Balázs

    2012-03-01

    Functional blood vasculature is essential for tumor progression. The main signalization pathways that play a key role in the survival and growth of tumor vessels originate from the VEGF-, PDGF- and FGF tyrosine kinase receptors. In the past decade, significant results have been published on receptor tyrosine kinase inhibitors (RTKIs). In this paper, the mechanisms of action and the results so far available of experimental and clinical studies on multi-target antiangiogenic TKIs are discussed. On the one hand, notable achievements have been made recently and these drugs are already used in clinical practice in some patient populations. On the other hand, the optimal combination and dosage of these drugs, selection of the apropriate biomarker and better understanding of the conflicting role of PDGFR and FGFR signaling in angiogenesis remain future challenges. PMID:22403757

  5. Improved Bearings-Only Multi-Target Tracking with GM-PHD Filtering

    PubMed Central

    Zhang, Qian; Song, Taek Lyul

    2016-01-01

    In this paper, an improved nonlinear Gaussian mixture probability hypothesis density (GM-PHD) filter is proposed to address bearings-only measurements in multi-target tracking. The proposed method, called the Gaussian mixture measurements-probability hypothesis density (GMM-PHD) filter, not only approximates the posterior intensity using a Gaussian mixture, but also models the likelihood function with a Gaussian mixture instead of a single Gaussian distribution. Besides, the target birth model of the GMM-PHD filter is assumed to be partially uniform instead of a Gaussian mixture. Simulation results show that the proposed filter outperforms the GM-PHD filter embedded with the extended Kalman filter (EKF) and the unscented Kalman filter (UKF). PMID:27626423

  6. Synthesis and pharmacological evaluation of piperidine (piperazine)-substituted benzoxazole derivatives as multi-target antipsychotics.

    PubMed

    Huang, Ling; Zhang, Wenjun; Zhang, Xiaohua; Yin, Lei; Chen, Bangyin; Song, Jinchun

    2015-11-15

    The present study describes the optimization of a series of novel benzoxazole-piperidine (piperazine) derivatives combining high dopamine D2 and serotonin 5-HT1A, 5-HT2A receptor affinities. Of these derivatives, the pharmacological features of compound 29 exhibited high affinities for the DA D2, 5-HT1A and 5-HT2A receptors, but low affinities for the 5-HT2C and histamine H1 receptors and human ether-a-go-go-related gene (hERG) channels. Furthermore, compound 29 reduced apomorphine-induced climbing and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head twitching without observable catalepsy, even at the highest dose tested. Thus, compound 29 is a promising candidate as a multi-target antipsychotic treatment.

  7. Multi-Target Detection from Full-Waveform Airborne Laser Scanner Using Phd Filter

    NASA Astrophysics Data System (ADS)

    Fuse, T.; Hiramatsu, D.; Nakanishi, W.

    2016-06-01

    We propose a new technique to detect multiple targets from full-waveform airborne laser scanner. We introduce probability hypothesis density (PHD) filter, a type of Bayesian filtering, by which we can estimate the number of targets and their positions simultaneously. PHD filter overcomes some limitations of conventional Gaussian decomposition method; PHD filter doesn't require a priori knowledge on the number of targets, assumption of parametric form of the intensity distribution. In addition, it can take a similarity between successive irradiations into account by modelling relative positions of the same targets spatially. Firstly we explain PHD filter and particle filter implementation to it. Secondly we formulate the multi-target detection problem on PHD filter by modelling components and parameters within it. At last we conducted the experiment on real data of forest and vegetation, and confirmed its ability and accuracy.

  8. AVN-101: A Multi-Target Drug Candidate for the Treatment of CNS Disorders

    PubMed Central

    Ivachtchenko, Alexandre V.; Lavrovsky, Yan; Okun, Ilya

    2016-01-01

    Lack of efficacy of many new highly selective and specific drug candidates in treating diseases with poorly understood or complex etiology, as are many of central nervous system (CNS) diseases, encouraged an idea of developing multi-modal (multi-targeted) drugs. In this manuscript, we describe molecular pharmacology, in vitro ADME, pharmacokinetics in animals and humans (part of the Phase I clinical studies), bio-distribution, bioavailability, in vivo efficacy, and safety profile of the multimodal drug candidate, AVN-101. We have carried out development of a next generation drug candidate with a multi-targeted mechanism of action, to treat CNS disorders. AVN-101 is a very potent 5-HT7 receptor antagonist (Ki = 153 pM), with slightly lesser potency toward 5-HT6, 5-HT2A, and 5HT-2C receptors (Ki = 1.2–2.0 nM). AVN-101 also exhibits a rather high affinity toward histamine H1 (Ki = 0.58 nM) and adrenergic α2A, α2B, and α2C (Ki = 0.41–3.6 nM) receptors. AVN-101 shows a good oral bioavailability and facilitated brain-blood barrier permeability, low toxicity, and reasonable efficacy in animal models of CNS diseases. The Phase I clinical study indicates the AVN-101 to be well tolerated when taken orally at doses of up to 20 mg daily. It does not dramatically influence plasma and urine biochemistry, nor does it prolong QT ECG interval, thus indicating low safety concerns. The primary therapeutic area for AVN-101 to be tested in clinical trials would be Alzheimer’s disease. However, due to its anxiolytic and anti-depressive activities, there is a strong rational for it to also be studied in such diseases as general anxiety disorders, depression, schizophrenia, and multiple sclerosis. PMID:27232215

  9. Promises of novel multi-target neuroprotective and neurorestorative drugs for Parkinson's disease.

    PubMed

    Youdim, Moussa B H; Kupershmidt, Lana; Amit, Tamar; Weinreb, Orly

    2014-01-01

    The cascade of neurotoxic events involved in neuronal degeneration suggests that it is naive to think mono-target drugs can induce disease modification by slowing the process of neurodegeneration in Parkinson's disease (PD). Employing the pharmacophore of rasagiline (N-propargyl-1-R-aminoindan), we have developed a series of novel multi-target neuroprotective drugs, including: (A) drugs [ladostigil, TV-3326 (N-propargyl-3R-aminoindan-5yl)-ethyl methylcarbamate)] with both cholinesterase-butyrylesterase (Ch-BuE) and brain-selective monamine oxidase-AB (MAO-AB) inhibitory activities and (B) iron chelator-radical scavenging drugs (M30) possessing brain-selective MAO-AB inhibitor activity and the neuroprotective-neurorescue propargylamine moiety of rasagiline. This was considered to be valid since brain MAO and iron increase in PD and aging, which could lead to oxidative stress-dependent neurodegeneration. The multi-target iron chelator, M30, has all the properties of ladostigil, but is not an acetylcholinesterase (CHE) inhibitor. However, M30 has both neuroprotective and neurorestorative activities for nigrostriatal dopamine neurons in post-lesion MPTP, lactacystin and 6-hydroxydopamine animal models of PD. The neurorestorative activity has been identified as being related to the ability of the drug to activate hypoxia-inducible factor (HIF) by inhibiting prolyl-4-hydroxylase. M30 regulates cell cycle arrest and induces the neurotrophins brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), erythropoietin (EPO), as well as glia-derived neurotrophic factor (GDNF). These unique multiple actions of M30 make it potentially useful as a disease modifying drug for the treatment of PD. PMID:24262165

  10. AVN-101: A Multi-Target Drug Candidate for the Treatment of CNS Disorders.

    PubMed

    Ivachtchenko, Alexandre V; Lavrovsky, Yan; Okun, Ilya

    2016-05-25

    Lack of efficacy of many new highly selective and specific drug candidates in treating diseases with poorly understood or complex etiology, as are many of central nervous system (CNS) diseases, encouraged an idea of developing multi-modal (multi-targeted) drugs. In this manuscript, we describe molecular pharmacology, in vitro ADME, pharmacokinetics in animals and humans (part of the Phase I clinical studies), bio-distribution, bioavailability, in vivo efficacy, and safety profile of the multimodal drug candidate, AVN-101. We have carried out development of a next generation drug candidate with a multi-targeted mechanism of action, to treat CNS disorders. AVN-101 is a very potent 5-HT7 receptor antagonist (Ki = 153 pM), with slightly lesser potency toward 5-HT6, 5-HT2A, and 5HT-2C receptors (Ki = 1.2-2.0 nM). AVN-101 also exhibits a rather high affinity toward histamine H1 (Ki = 0.58 nM) and adrenergic α2A, α2B, and α2C (Ki = 0.41-3.6 nM) receptors. AVN-101 shows a good oral bioavailability and facilitated brain-blood barrier permeability, low toxicity, and reasonable efficacy in animal models of CNS diseases. The Phase I clinical study indicates the AVN-101 to be well tolerated when taken orally at doses of up to 20 mg daily. It does not dramatically influence plasma and urine biochemistry, nor does it prolong QT ECG interval, thus indicating low safety concerns. The primary therapeutic area for AVN-101 to be tested in clinical trials would be Alzheimer's disease. However, due to its anxiolytic and anti-depressive activities, there is a strong rational for it to also be studied in such diseases as general anxiety disorders, depression, schizophrenia, and multiple sclerosis. PMID:27232215

  11. [Development of multi-target multi-spectral high-speed pyrometer].

    PubMed

    Xiao, Peng; Dai, Jing-Min; Wang, Qing-Wei

    2008-11-01

    The plume temperature of a solid propellant rocket engine (SPRE) is a fundamental parameter in denoting combustion status. It is necessary to measure the temperature along both the axis and the radius of the engine. In order to measure the plume temperature distribution of a solid propellant rocket engine, the multi-spectral thermometry has been approved. Previously the pyrometer was developed in the Harbin Institute of Technology of China in 1999, which completed the measurement of SPRE plume temperature and its distribution with multi-spectral technique in aerospace model development for the first time. Following this experience, a new type of multi-target multi-spectral high-speed pyrometer used in the ground experiments of SPRE plume temperature measurement was developed. The main features of the instrument include the use of a dispersing prism and a photo-diode array to cover the entire spectral band of 0.4 to 1.1 microm. The optic fibers are used in order to collect and transmit the thermal radiation fluxes. The instrument can measure simultaneously the temperature and emissivity of eight spectra for six uniformly distributed points on the target surface, which are well defined by the hole on the field stop lens. A specially designed S/H (Sample/Hold) circuit, with 48 sample and hold units that were triggered with a signal, measures the multi-spectral and multi-target outputs. It can sample 48 signals with a less than 10ns time difference which is most important for the temperature calculation. PMID:19271529

  12. A novel square-root cubature information weighted consensus filter algorithm for multi-target tracking in distributed camera networks.

    PubMed

    Chen, Yanming; Zhao, Qingjie

    2015-01-01

    This paper deals with the problem of multi-target tracking in a distributed camera network using the square-root cubature information filter (SCIF). SCIF is an efficient and robust nonlinear filter for multi-sensor data fusion. In camera networks, multiple cameras are arranged in a dispersed manner to cover a large area, and the target may appear in the blind area due to the limited field of view (FOV). Besides, each camera might receive noisy measurements. To overcome these problems, this paper proposes a novel multi-target square-root cubature information weighted consensus filter (MTSCF), which reduces the effect of clutter or spurious measurements using joint probabilistic data association (JPDA) and proper weights on the information matrix and information vector. The simulation results show that the proposed algorithm can efficiently track multiple targets in camera networks and is obviously better in terms of accuracy and stability than conventional multi-target tracking algorithms. PMID:25951338

  13. A novel square-root cubature information weighted consensus filter algorithm for multi-target tracking in distributed camera networks.

    PubMed

    Chen, Yanming; Zhao, Qingjie

    2015-05-05

    This paper deals with the problem of multi-target tracking in a distributed camera network using the square-root cubature information filter (SCIF). SCIF is an efficient and robust nonlinear filter for multi-sensor data fusion. In camera networks, multiple cameras are arranged in a dispersed manner to cover a large area, and the target may appear in the blind area due to the limited field of view (FOV). Besides, each camera might receive noisy measurements. To overcome these problems, this paper proposes a novel multi-target square-root cubature information weighted consensus filter (MTSCF), which reduces the effect of clutter or spurious measurements using joint probabilistic data association (JPDA) and proper weights on the information matrix and information vector. The simulation results show that the proposed algorithm can efficiently track multiple targets in camera networks and is obviously better in terms of accuracy and stability than conventional multi-target tracking algorithms.

  14. A Novel Square-Root Cubature Information Weighted Consensus Filter Algorithm for Multi-Target Tracking in Distributed Camera Networks

    PubMed Central

    Chen, Yanming; Zhao, Qingjie

    2015-01-01

    This paper deals with the problem of multi-target tracking in a distributed camera network using the square-root cubature information filter (SCIF). SCIF is an efficient and robust nonlinear filter for multi-sensor data fusion. In camera networks, multiple cameras are arranged in a dispersed manner to cover a large area, and the target may appear in the blind area due to the limited field of view (FOV). Besides, each camera might receive noisy measurements. To overcome these problems, this paper proposes a novel multi-target square-root cubature information weighted consensus filter (MTSCF), which reduces the effect of clutter or spurious measurements using joint probabilistic data association (JPDA) and proper weights on the information matrix and information vector. The simulation results show that the proposed algorithm can efficiently track multiple targets in camera networks and is obviously better in terms of accuracy and stability than conventional multi-target tracking algorithms. PMID:25951338

  15. Elucidation of gene function using C-5 propyne antisense oligonucleotides.

    PubMed

    Flanagan, W M; Su, L L; Wagner, R W

    1996-09-01

    Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells. PMID:9631067

  16. The role of antisense oligonucleotide therapy in patients with familial hypercholesterolemia: risks, benefits, and management recommendations.

    PubMed

    Agarwala, Anandita; Jones, Peter; Nambi, Vijay

    2015-01-01

    Antisense oligonucleotide therapy is a promising approach for the treatment of a broad variety of medical conditions. It functions at the cellular level by interfering with RNA function, often leading to degradation of specifically targeted abnormal gene products implicated in the disease process. Mipomersen is a novel antisense oligonucleotide directed at apolipoprotein (apoB)-100, the primary apolipoprotein associated with low-density lipoprotein cholesterol (LDL-C), which has recently been approved for the treatment of familial hypercholesterolemia. A number of clinical studies have demonstrated its efficacy in lowering LDL-C and apoB levels in patients with elevated LDL-C despite maximal medical therapy using conventional lipid-lowering agents. This review outlines the risks and benefits of therapy and provides recommendations on the use of mipomersen.

  17. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    PubMed

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

    2014-01-01

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

  18. Undetected antisense tRNAs in mitochondrial genomes?

    PubMed Central

    2010-01-01

    Background The hypothesis that both mitochondrial (mt) complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding) is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and Dr Andrei S Rodin (nominated

  19. Antisense oligonucleotides, microRNAs, and antibodies.

    PubMed

    Dávalos, Alberto; Chroni, Angeliki

    2015-01-01

    The specificity of Watson-Crick base pairing and the development of several chemical modifications to oligonucleotides have enabled the development of novel drug classes for the treatment of different human diseases. This review focuses on promising results of recent preclinical or clinical studies on targeting HDL metabolism and function by antisense oligonucleotides and miRNA-based therapies. Although many hurdles regarding basic mechanism of action, delivery, specificity, and toxicity need to be overcome, promising results from recent clinical trials and recent approval of these types of therapy to treat dyslipidemia suggest that the treatment of HDL dysfunction will benefit from these unique clinical opportunities. Moreover, an overview of monoclonal antibodies (mAbs) developed for the treatment of dyslipidemia and cardiovascular disease and currently being tested in clinical studies is provided. Initial studies have shown that these compounds are generally safe and well tolerated, but ongoing large clinical studies will assess their long-term safety and efficacy.

  20. Design, synthesis and evaluation of seleno-dihydropyrimidinones as potential multi-targeted therapeutics for Alzheimer's disease.

    PubMed

    Canto, Rômulo F S; Barbosa, Flavio A R; Nascimento, Vanessa; de Oliveira, Aldo S; Brighente, Inês M C; Braga, Antonio Luiz

    2014-06-01

    In this paper we report the design, synthesis and evaluation of a series of seleno-dihydropyrimidinones as potential multi-targeted therapeutics for Alzheimer's disease. The compounds show excellent results as acetylcholinesterase inhibitors, being as active as the standard drug. All these compounds also show very good antioxidant activity through different mechanisms of action.

  1. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  2. Topology of classical molecular optimal control landscapes for multi-target objectives

    NASA Astrophysics Data System (ADS)

    Joe-Wong, Carlee; Ho, Tak-San; Rabitz, Herschel; Wu, Rebing

    2015-04-01

    This paper considers laser-driven optimal control of an ensemble of non-interacting molecules whose dynamics lie in classical phase space. The molecules evolve independently under control to distinct final states. We consider a control landscape defined in terms of multi-target (MT) molecular states and analyze the landscape as a functional of the control field. The topology of the MT control landscape is assessed through its gradient and Hessian with respect to the control. Under particular assumptions, the MT control landscape is found to be free of traps that could hinder reaching the objective. The Hessian associated with an optimal control field is shown to have finite rank, indicating an inherent degree of robustness to control noise. Both the absence of traps and rank of the Hessian are shown to be analogous to the situation of specifying multiple targets for an ensemble of quantum states. Numerical simulations are presented to illustrate the classical landscape principles and further characterize the system behavior as the control field is optimized.

  3. Antenna Allocation in MIMO Radar with Widely Separated Antennas for Multi-Target Detection

    PubMed Central

    Gao, Hao; Wang, Jian; Jiang, Chunxiao; Zhang, Xudong

    2014-01-01

    In this paper, we explore a new resource called multi-target diversity to optimize the performance of multiple input multiple output (MIMO) radar with widely separated antennas for detecting multiple targets. In particular, we allocate antennas of the MIMO radar to probe different targets simultaneously in a flexible manner based on the performance metric of relative entropy. Two antenna allocation schemes are proposed. In the first scheme, each antenna is allocated to illuminate a proper target over the entire illumination time, so that the detection performance of each target is guaranteed. The problem is formulated as a minimum makespan scheduling problem in the combinatorial optimization framework. Antenna allocation is implemented through a branch-and-bound algorithm and an enhanced factor 2 algorithm. In the second scheme, called antenna-time allocation, each antenna is allocated to illuminate different targets with different illumination time. Both antenna allocation and time allocation are optimized based on illumination probabilities. Over a large range of transmitted power, target fluctuations and target numbers, both of the proposed antenna allocation schemes outperform the scheme without antenna allocation. Moreover, the antenna-time allocation scheme achieves a more robust detection performance than branch-and-bound algorithm and the enhanced factor 2 algorithm when the target number changes. PMID:25350505

  4. Antenna allocation in MIMO radar with widely separated antennas for multi-target detection.

    PubMed

    Gao, Hao; Wang, Jian; Jiang, Chunxiao; Zhang, Xudong

    2014-10-27

    In this paper, we explore a new resource called multi-target diversity to optimize the performance of multiple input multiple output (MIMO) radar with widely separated antennas for detecting multiple targets. In particular, we allocate antennas of the MIMO radar to probe different targets simultaneously in a flexible manner based on the performance metric of relative entropy. Two antenna allocation schemes are proposed. In the first scheme, each antenna is allocated to illuminate a proper target over the entire illumination time, so that the detection performance of each target is guaranteed. The problem is formulated as a minimum makespan scheduling problem in the combinatorial optimization framework. Antenna allocation is implemented through a branch-and-bound algorithm and an enhanced factor 2 algorithm. In the second scheme, called antenna-time allocation, each antenna is allocated to illuminate different targets with different illumination time. Both antenna allocation and time allocation are optimized based on illumination probabilities. Over a large range of transmitted power, target fluctuations and target numbers, both of the proposed antenna allocation schemes outperform the scheme without antenna allocation. Moreover, the antenna-time allocation scheme achieves a more robust detection performance than branch-and-bound algorithm and the enhanced factor 2 algorithm when the target number changes.

  5. Gait measurement system for the multi-target stepping task using a laser range sensor.

    PubMed

    Yorozu, Ayanori; Nishiguchi, Shu; Yamada, Minoru; Aoyama, Tomoki; Moriguchi, Toshiki; Takahashi, Masaki

    2015-01-01

    For the prevention of falling in the elderly, gait training has been proposed using tasks such as the multi-target stepping task (MTST), in which participants step on assigned colored targets. This study presents a gait measurement system using a laser range sensor for the MTST to evaluate the risk of falling. The system tracks both legs and measures general walking parameters such as stride length and walking speed. Additionally, it judges whether the participant steps on the assigned colored targets and detects cross steps to evaluate cognitive function. However, situations in which one leg is hidden from the sensor or the legs are close occur and are likely to lead to losing track of the legs or false tracking. To solve these problems, we propose a novel leg detection method with five observed leg patterns and global nearest neighbor-based data association with a variable validation region based on the state of each leg. In addition, methods to judge target steps and detect cross steps based on leg trajectory are proposed. From the experimental results with the elderly, it is confirmed that the proposed system can improve leg-tracking performance, judge target steps and detect cross steps with high accuracy. PMID:25985161

  6. Multi-target camera tracking, hand-off and display LDRD 158819 final report

    SciTech Connect

    Anderson, Robert J.

    2014-10-01

    Modern security control rooms gather video and sensor feeds from tens to hundreds of cameras. Advanced camera analytics can detect motion from individual video streams and convert unexpected motion into alarms, but the interpretation of these alarms depends heavily upon human operators. Unfortunately, these operators can be overwhelmed when a large number of events happen simultaneously, or lulled into complacency due to frequent false alarms. This LDRD project has focused on improving video surveillance-based security systems by changing the fundamental focus from the cameras to the targets being tracked. If properly integrated, more cameras shouldn't lead to more alarms, more monitors, more operators, and increased response latency but instead should lead to better information and more rapid response times. For the course of the LDRD we have been developing algorithms that takes live video imagery from multiple video cameras, identifies individual moving targets from the background imagery, and then displays the results in a single 3D interactive video. In this document we summarize the work in developing this multi-camera, multi-target system, including lessons learned, tools developed, technologies explored, and a description of currently capability.

  7. Multi-Target Camera Tracking, Hand-off and Display LDRD 158819 Final Report

    SciTech Connect

    Anderson, Robert J.

    2014-10-01

    Modern security control rooms gather video and sensor feeds from tens to hundreds of cameras. Advanced camera analytics can detect motion from individual video streams and convert unexpected motion into alarms, but the interpretation of these alarms depends heavily upon human operators. Unfortunately, these operators can be overwhelmed when a large number of events happen simultaneously, or lulled into complacency due to frequent false alarms. This LDRD project has focused on improving video surveillance based security systems by changing the fundamental focus from the cameras to the targets being tracked. If properly integrated, more cameras shouldn’t lead to more alarms, more monitors, more operators, and increased response latency but instead should lead to better information and more rapid response times. For the course of the LDRD we have been developing algorithms that takes live video imagery from multiple video cameras, identifies individual moving targets from the background imagery, and then displays the results in a single 3D interactive video. In this document we summarize the work in developing this multi-camera, multi-target system, including lessons learned, tools developed, technologies explored, and a description of currently capability.

  8. Gait measurement system for the multi-target stepping task using a laser range sensor.

    PubMed

    Yorozu, Ayanori; Nishiguchi, Shu; Yamada, Minoru; Aoyama, Tomoki; Moriguchi, Toshiki; Takahashi, Masaki

    2015-05-13

    For the prevention of falling in the elderly, gait training has been proposed using tasks such as the multi-target stepping task (MTST), in which participants step on assigned colored targets. This study presents a gait measurement system using a laser range sensor for the MTST to evaluate the risk of falling. The system tracks both legs and measures general walking parameters such as stride length and walking speed. Additionally, it judges whether the participant steps on the assigned colored targets and detects cross steps to evaluate cognitive function. However, situations in which one leg is hidden from the sensor or the legs are close occur and are likely to lead to losing track of the legs or false tracking. To solve these problems, we propose a novel leg detection method with five observed leg patterns and global nearest neighbor-based data association with a variable validation region based on the state of each leg. In addition, methods to judge target steps and detect cross steps based on leg trajectory are proposed. From the experimental results with the elderly, it is confirmed that the proposed system can improve leg-tracking performance, judge target steps and detect cross steps with high accuracy.

  9. Joint decision and Naive Bayes learning for detection of space multi-target

    NASA Astrophysics Data System (ADS)

    Huang, Tao; Li, Zhulian; Zhou, Yu; Xiong, Yaoheng; Zhang, Haitao

    2014-07-01

    In the photoelectric tracking system, the detection of space multi-target is crucial for target localization and tracking. The difficulties include the interferences from CCD smear and strong noise, the few characteristics of spot-like targets and the challenge of multiple targets. In this paper, we propose a hybrid algorithm of joint decision and Naive Bayes (JD-NB) learning, and present the duty ratio feature to discriminate the target and smear blocks. Firstly, we extract the proper features and train the parameters of the Naive Bayes classifier. Secondly, target blocks are preliminarily estimated with the Naive Bayes. Lastly, the 4-adjacent blocks of the candidate target blocks are jointed to analyze the distribution pattern and the true target blocks are secondarily extracted by the method of pattern matching. Experimental results indicate that the proposed JD-NB algorithm not only possesses a high recognition rate of better than 90% for the target block, but also effectively overcomes the disturbance of the smear block. Moreover, it performs well in the detection of small and faint targets when the SNR of the block is higher than about 0.014.

  10. Gait Measurement System for the Multi-Target Stepping Task Using a Laser Range Sensor

    PubMed Central

    Yorozu, Ayanori; Nishiguchi, Shu; Yamada, Minoru; Aoyama, Tomoki; Moriguchi, Toshiki; Takahashi, Masaki

    2015-01-01

    For the prevention of falling in the elderly, gait training has been proposed using tasks such as the multi-target stepping task (MTST), in which participants step on assigned colored targets. This study presents a gait measurement system using a laser range sensor for the MTST to evaluate the risk of falling. The system tracks both legs and measures general walking parameters such as stride length and walking speed. Additionally, it judges whether the participant steps on the assigned colored targets and detects cross steps to evaluate cognitive function. However, situations in which one leg is hidden from the sensor or the legs are close occur and are likely to lead to losing track of the legs or false tracking. To solve these problems, we propose a novel leg detection method with five observed leg patterns and global nearest neighbor-based data association with a variable validation region based on the state of each leg. In addition, methods to judge target steps and detect cross steps based on leg trajectory are proposed. From the experimental results with the elderly, it is confirmed that the proposed system can improve leg-tracking performance, judge target steps and detect cross steps with high accuracy. PMID:25985161

  11. Topology of classical molecular optimal control landscapes for multi-target objectives

    SciTech Connect

    Joe-Wong, Carlee; Ho, Tak-San; Rabitz, Herschel; Wu, Rebing

    2015-04-21

    This paper considers laser-driven optimal control of an ensemble of non-interacting molecules whose dynamics lie in classical phase space. The molecules evolve independently under control to distinct final states. We consider a control landscape defined in terms of multi-target (MT) molecular states and analyze the landscape as a functional of the control field. The topology of the MT control landscape is assessed through its gradient and Hessian with respect to the control. Under particular assumptions, the MT control landscape is found to be free of traps that could hinder reaching the objective. The Hessian associated with an optimal control field is shown to have finite rank, indicating an inherent degree of robustness to control noise. Both the absence of traps and rank of the Hessian are shown to be analogous to the situation of specifying multiple targets for an ensemble of quantum states. Numerical simulations are presented to illustrate the classical landscape principles and further characterize the system behavior as the control field is optimized.

  12. In Vitro and In Vivo Activity of Multi-Target Inhibitors Against Trypanosoma brucei

    PubMed Central

    Yang, Gyongseon; Zhu, Wei; Wang, Yang; Huang, Guozhong; Byun, Sooyoung; Choi, Gahee; Li, Kai; Huang, Zhuoli; Docampo, Roberto; Oldfield, Eric; No, Joo Hwan

    2015-01-01

    We tested a series of amidine and related compounds against Trypanosoma brucei. The most active compound was a biphenyldiamidine which had an EC50 of 7.7 nM against bloodstream form parasites. There was little toxicity against two human cell lines with CC50 > 100 μM. There was also good in vivo activity in a mouse model of infection with 100% survival at 3 mg/kg i.p. The most potent lead blocked replication of kinetoplast DNA (k-DNA), but not nuclear DNA, in the parasite. Some compounds also inhibited the enzyme farnesyl diphosphate synthase (FPPS) and some were uncouplers of oxidative phosphorylation. We developed a computational model for T. brucei cell growth inhibition (R2 = 0.76) using DNA ΔTm values for inhibitor binding, combined with T. brucei FPPS IC50 values. Overall, the results suggest that it may be possible to develop multi-target drug leads against T. brucei that act by inhibiting both k-DNA replication and isoprenoid biosynthesis. PMID:26295062

  13. Topology of classical molecular optimal control landscapes for multi-target objectives.

    PubMed

    Joe-Wong, Carlee; Ho, Tak-San; Rabitz, Herschel; Wu, Rebing

    2015-04-21

    This paper considers laser-driven optimal control of an ensemble of non-interacting molecules whose dynamics lie in classical phase space. The molecules evolve independently under control to distinct final states. We consider a control landscape defined in terms of multi-target (MT) molecular states and analyze the landscape as a functional of the control field. The topology of the MT control landscape is assessed through its gradient and Hessian with respect to the control. Under particular assumptions, the MT control landscape is found to be free of traps that could hinder reaching the objective. The Hessian associated with an optimal control field is shown to have finite rank, indicating an inherent degree of robustness to control noise. Both the absence of traps and rank of the Hessian are shown to be analogous to the situation of specifying multiple targets for an ensemble of quantum states. Numerical simulations are presented to illustrate the classical landscape principles and further characterize the system behavior as the control field is optimized.

  14. Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen; Chatterjee, Anushree

    2014-03-01

    ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

  15. Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

    PubMed

    Roh, Young Hoon; Lee, Jong Bum; Shopsowitz, Kevin E; Dreaden, Erik C; Morton, Stephen W; Poon, Zhiyong; Hong, Jinkee; Yamin, Inbar; Bonner, Daniel K; Hammond, Paula T

    2014-10-28

    Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

  16. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    SciTech Connect

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

    2010-03-12

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  17. Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

    PubMed Central

    Mundodi, V; Kucknoor, AS; Alderete, JF

    2007-01-01

    Background Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. Results We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. Conclusion These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis. PMID:17608941

  18. NTZIP antisense plants show reduced chlorophyll levels.

    PubMed

    Liu, Ning; Yang, Yu-Tao; Liu, Han-Hua; Yang, Guo-Dong; Zhang, Nai-Hua; Zheng, Cheng Chao

    2004-04-01

    We have isolated and characterized a new photosynthetic tissue-specific gene NTZIP (Nicotiana tabacum leucine zipper) from tobacco (N. tabacum). Its deduced amino acid sequence has two highly conserved regions, leucine zipper and [EX(n)DEXRH](2) motifs, which are related to the gene's biochemical functions. NTZIP was expressed in leaves and stems, but was not detected in roots or flowers, suggesting that its physiological functions might be associated with photosynthesis. Northern blot analysis showed that NTZIP mRNA accumulation was induced by light signals, increased greatly under low temperatures and was repressed by strong light illumination. Furthermore, a number of homologs of NTZIP were isolated from cucumber (Cucumis sativus), rape (Brassica napus), clover (Trifolium repens), willow (Salix babylonica), rosebush (Rusa dovurica), wheat (Triticum aestivum) and spinach (Spinacia oleracea), proving the ubiquitous existence of the NTZIP-like genes in higher plants. Transgenic tobaccos constitutively expressing antisense RNA to NTZIP displayed chlorosis and a lack of ability to turn green even under normal growth conditions. The chlorophyll deficiency was further confirmed by chlorophyll content determination and gas exchange analysis. Based on these observations, we propose that NTZIP may be involved in chlorophyll biosynthesis, and might define a novel family of evolutionarily conserved proteins with its homologs in other plant species.

  19. Olive Oil Phenols as Promising Multi-targeting Agents Against Alzheimer's Disease.

    PubMed

    Rigacci, Stefania

    2015-01-01

    Amyloid diseases are characterized by the deposition of typically aggregated proteins/peptides in tissues, associated with degeneration and progressive functional impairment. Alzheimer's disease is one of the most studied neurodegenerative amyloid diseases and, in Western countries, a significant cause of dementia in the elderly. The so-called "Mediterranean diet" has been considered for long as the healthier dietary regimen, characterised by a great abundance in vegetables and fruits, extra virgin olive oil as the main source of fat, a moderate consumption of red wine and a reduced intake of proteins from red meat. Recent epidemiological studies support the efficacy of the Mediterranean diet not only against cardiovascular and cancer diseases (as previously demonstrated) but also against the cognitive decline associated with ageing, and several data are highlighting the role played by natural phenols, of which red wine and extra virgin olive oil are rich, in such context. In the meantime, studies conducted both in vivo and in vitro have started to reveal the great potential of the phenolic component of extra virgin olive oil (mainly oleuropein aglycone and oleocanthal) in counteracting amyloid aggregation and toxicity, with a particular emphasis on the pathways involved in the onset and progression of Alzheimer's disease: amyloid precursor protein processing, amyloid-beta (Aβ) peptide and tau aggregation, autophagy impairment, neuroinflammation. The aim of this review is to summarize the results of such research efforts, showing how the action of these phenols goes far beyond their renowned antioxidant activity and revealing their potential as multi-targeting agents against Alzheimer's disease. PMID:26092624

  20. Piezo-microfluidic transport system for multi-targets biochip detections

    NASA Astrophysics Data System (ADS)

    Li, Chia-Chin; Wang, Pei-Wen; Lee, Chih-Kung

    2016-03-01

    Detecting minute trace of interferon-gamma and various bio-markers by using a single biochip was adopted as a platform to examine the technology advancements presented. As bio-detection faces the restriction that only very small quantity of specimen is available, ways to make the best use of the sample available are a must. Since samples concentration will affect the binding rate of an immunoassay, the testing order will become an influencing factor if multiple biomarkers testing situation are needed by using only a single trace of sample. More specifically, if we test disease A first and then detect disease B using the sample just been measured by testing disease A, we most likely will get different results if we reverse the testing order. With an attempt to examine and maybe resolve the issues mentioned above, a micro-fluid control system was developed. The design requirements not only ask for microfluidic control but also demand the system developed has the potential to be integrated within the biochip once its performance is verified. A piezo-vibrating system that can generate traveling waves for microfluidic control was chosen due to its versatility and large force to volume ratio. A simulation software COMSOL was adopted first to predict the microfluidic behavior of the two-mode excited piezo-microfluidic transport system. Secondly, fluorescent particles was used to analyze the microfluidic behavior of system fabricated based on the simulation. Finally, Electrochemistry Impedance Spectroscopy (EIS) was implemented to verify the performance and extendibility of this newly developed system for multi-target detections.

  1. Olive Oil Phenols as Promising Multi-targeting Agents Against Alzheimer's Disease.

    PubMed

    Rigacci, Stefania

    2015-01-01

    Amyloid diseases are characterized by the deposition of typically aggregated proteins/peptides in tissues, associated with degeneration and progressive functional impairment. Alzheimer's disease is one of the most studied neurodegenerative amyloid diseases and, in Western countries, a significant cause of dementia in the elderly. The so-called "Mediterranean diet" has been considered for long as the healthier dietary regimen, characterised by a great abundance in vegetables and fruits, extra virgin olive oil as the main source of fat, a moderate consumption of red wine and a reduced intake of proteins from red meat. Recent epidemiological studies support the efficacy of the Mediterranean diet not only against cardiovascular and cancer diseases (as previously demonstrated) but also against the cognitive decline associated with ageing, and several data are highlighting the role played by natural phenols, of which red wine and extra virgin olive oil are rich, in such context. In the meantime, studies conducted both in vivo and in vitro have started to reveal the great potential of the phenolic component of extra virgin olive oil (mainly oleuropein aglycone and oleocanthal) in counteracting amyloid aggregation and toxicity, with a particular emphasis on the pathways involved in the onset and progression of Alzheimer's disease: amyloid precursor protein processing, amyloid-beta (Aβ) peptide and tau aggregation, autophagy impairment, neuroinflammation. The aim of this review is to summarize the results of such research efforts, showing how the action of these phenols goes far beyond their renowned antioxidant activity and revealing their potential as multi-targeting agents against Alzheimer's disease.

  2. Imbricaric Acid and Perlatolic Acid: Multi-Targeting Anti-Inflammatory Depsides from Cetrelia monachorum

    PubMed Central

    Oettl, Sarah K.; Gerstmeier, Jana; Khan, Shafaat Y.; Wiechmann, Katja; Bauer, Julia; Atanasov, Atanas G.; Malainer, Clemens; Awad, Ezzat M.; Uhrin, Pavel; Heiss, Elke H.; Waltenberger, Birgit; Remias, Daniel; Breuss, Johannes M.; Boustie, Joel; Dirsch, Verena M.; Stuppner, Hermann; Werz, Oliver; Rollinger, Judith M.

    2013-01-01

    In vitro screening of 17 Alpine lichen species for their inhibitory activity against 5-lipoxygenase, microsomal prostaglandin E2 synthase-1 and nuclear factor kappa B revealed Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. As conceivable source for novel anti-inflammatory compounds. Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid (4) and perlatolic acid (5) approved dual inhibitory activities on microsomal prostaglandin E2 synthase-1 (IC50 = 1.9 and 0.4 µM, resp.) and on 5-lipoxygenase tested in a cell-based assay (IC50 = 5.3 and 1.8 µM, resp.) and on purified enzyme (IC50 = 3.5 and 0.4 µM, resp.). Additionally, these two main constituents quantified in the extract with 15.22% (4) and 9.10% (5) showed significant inhibition of tumor necrosis factor alpha-induced nuclear factor kappa B activation in luciferase reporter cells with IC50 values of 2.0 and 7.0 µM, respectively. In a murine in vivo model of inflammation, 5 impaired the inflammatory, thioglycollate-induced recruitment of leukocytes to the peritoneum. The potent inhibitory effects on the three identified targets attest 4 and 5 a pronounced multi-target anti-inflammatory profile which warrants further investigation on their pharmacokinetics and in vivo efficacy. PMID:24130812

  3. The Multi-Target Drug M30 Shows Pro-Cognitive and Anti-Inflammatory Effects in a Rat Model of Alzheimer's Disease.

    PubMed

    Pimentel, Luisa S; Allard, Simon; Do Carmo, Sonia; Weinreb, Orly; Danik, Marc; Hanzel, Cecilia E; Youdim, Moussa B; Cuello, A Claudio

    2015-01-01

    Current therapies for Alzheimer's disease (AD) offer partial symptomatic relief and do not modify disease progression. There is substantial evidence indicating a disease onset years before clinical diagnosis, at which point no effective therapy has been found. In this study, we investigated the efficacy of a new multi-target drug, M30, at relatively early stages of the AD-like amyloid pathology in a robust rat transgenic model. McGill-R-Thy1-APP transgenic rats develop the full AD-like amyloid pathology in a progressive fashion, and have a minimal genetic burden. McGill rats were given 5 mg/kg M30 or vehicle per os, every 2 days for 4 months, starting at a stage where the transgenic animals suffer detectable cognitive impairments. At the completion of the treatment, cognitive functions were assessed with Novel Object Location and Novel Object Recognition tests. The brains were then analyzed to assess amyloid-β (Aβ) burden and the levels of key inflammatory markers. Long-term treatment with M30 was associated with both the prevention and the reversal of transgene-related cognitive decline. The effects on cognition were accompanied by a shift of the Aβ-immunoreactive material toward an amyloid plaque aggregated molecular form, diminished molecular signs of CNS inflammation and a change in microglia morphology toward a surveying phenotype. This study is the first to demonstrate the therapeutic potential of M30 in a rat model of the AD amyloid pathology. It provides a rationale for further investigations with M30 and with potential multi-target approaches to delay, prevent or reverse the progression the AD pathology at early disease-stages. PMID:26401560

  4. Experimental demonstration of a multi-target detection technique using an X-band optically steered phased array radar.

    PubMed

    Shi, Nuannuan; Li, Ming; Deng, Ye; Zhang, Lihong; Sun, Shuqian; Tang, Jian; Li, Wei; Zhu, Ninghua

    2016-06-27

    An X-band optically-steered phased array radar is developed to demonstrate high resolution multi-target detection. The beam forming is implemented based on wavelength-swept true time delay (TTD) technique. The beam forming system has a wide direction tuning range of ± 54 degree, low magnitude ripple of ± 0.5 dB and small delay error of 0.13 ps/nm. To further verify performance of the proposed optically-steered phased array radar, three experiments are then carried out to implement the single and multiple target detection. A linearly chirped X-band microwave signal is used as radar signal which is finally compressed at the receiver to improve the detection accuracy. The ranging resolution for multi-target detection is up to 2 cm within the measuring distance over 4 m and the azimuth angle error is less than 4 degree.

  5. Experimental demonstration of a multi-target detection technique using an X-band optically steered phased array radar.

    PubMed

    Shi, Nuannuan; Li, Ming; Deng, Ye; Zhang, Lihong; Sun, Shuqian; Tang, Jian; Li, Wei; Zhu, Ninghua

    2016-06-27

    An X-band optically-steered phased array radar is developed to demonstrate high resolution multi-target detection. The beam forming is implemented based on wavelength-swept true time delay (TTD) technique. The beam forming system has a wide direction tuning range of ± 54 degree, low magnitude ripple of ± 0.5 dB and small delay error of 0.13 ps/nm. To further verify performance of the proposed optically-steered phased array radar, three experiments are then carried out to implement the single and multiple target detection. A linearly chirped X-band microwave signal is used as radar signal which is finally compressed at the receiver to improve the detection accuracy. The ranging resolution for multi-target detection is up to 2 cm within the measuring distance over 4 m and the azimuth angle error is less than 4 degree. PMID:27410597

  6. RNA therapeutics: RNAi and antisense mechanisms and clinical applications

    PubMed Central

    Chery, Jessica

    2016-01-01

    RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology1. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials2. Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs. PMID:27570789

  7. Antisense therapy for cancer--the time of truth.

    PubMed

    Jansen, Burkhard; Zangemeister-Wittke, Uwe

    2002-11-01

    The recent acceleration in the identification and characterisation of new molecular targets for cancer and the limited effectiveness of conventional treatment strategies has focused considerable interest on the development of new types of anticancer agents. These new drugs are hoped to be highly specific for malignant cells with a favorable side-effect profile due to well-defined mechanisms of action. Antisense oligonucleotides are one such class of new agent--they are short, synthetic stretches of DNA which hybridise with specific mRNA strands that correspond to target genes. By binding to the mRNA, the antisense oligonucleotides prevent the sequence of the target gene being converted into a protein, thereby blocking the action of the gene. Several genes known to be important in the regulation of apoptosis, cell growth, metastasis, and angiogenesis, have been validated as molecular targets for antisense therapy. Furthermore, new targets are rapidly being uncovered through coordinated functional genomics and proteomics initiatives. Phosphorothioate oligonucleotides are the current gold standard for antisense therapy; they have acceptable physical and chemical properties and show reasonable resistance to nucleases. Recently, new generations of these phosphorothioate oligonucleotides that contain 2'-modified nucleoside building blocks to enhance RNA binding affinity and decrease indirect toxic effects have been developed. Antisense therapeutics are, after decades of difficulties, finally close to fulfilling their promise in the clinic.

  8. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

    PubMed Central

    Bielinska, A; Kukowska-Latallo, J F; Johnson, J; Tomalia, D A; Baker, J R

    1996-01-01

    Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression. PMID:8668551

  9. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  10. Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility.

    PubMed

    Thipyapong, Piyada; Hunt, Michelle D; Steffens, John C

    2004-11-01

    Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm(-2) than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm(-2) than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::beta-glucuronidase constructs when plants are challenged with P

  11. Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA

    PubMed Central

    Player, Mark R.; Barnard, Dale L.; Torrence, Paul F.

    1998-01-01

    The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication. PMID:9671772

  12. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    PubMed

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  13. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  14. Multi-Target Directed Donepezil-Like Ligands for Alzheimer's Disease.

    PubMed

    Unzeta, Mercedes; Esteban, Gerard; Bolea, Irene; Fogel, Wieslawa A; Ramsay, Rona R; Youdim, Moussa B H; Tipton, Keith F; Marco-Contelles, José

    2016-01-01

    HIGHLIGHTS ASS234 is a MTDL compound containing a moiety from Donepezil and the propargyl group from the PF 9601N, a potent and selective MAO B inhibitor. This compound is the most advanced anti-Alzheimer agent for preclinical studies identified in our laboratory.Derived from ASS234 both multipotent donepezil-indolyl (MTDL-1) and donepezil-pyridyl hybrids (MTDL-2) were designed and evaluated as inhibitors of AChE/BuChE and both MAO isoforms. MTDL-2 showed more high affinity toward the four enzymes than MTDL-1.MTDL-3 and MTDL-4, were designed containing the N-benzylpiperidinium moiety from Donepezil, a metal- chelating 8-hydroxyquinoline group and linked to a N-propargyl core and they were pharmacologically evaluated.The presence of the cyano group in MTDL-3, enhanced binding to AChE, BuChE and MAO A. It showed antioxidant behavior and it was able to strongly complex Cu(II), Zn(II) and Fe(III).MTDL-4 showed higher affinity toward AChE, BuChE.MTDL-3 exhibited good brain penetration capacity (ADMET) and less toxicity than Donepezil. Memory deficits in scopolamine-lesioned animals were restored by MTDL-3.MTDL-3 particularly emerged as a ligand showing remarkable potential benefits for its use in AD therapy. Alzheimer's disease (AD), the most common form of adult onset dementia, is an age-related neurodegenerative disorder characterized by progressive memory loss, decline in language skills, and other cognitive impairments. Although its etiology is not completely known, several factors including deficits of acetylcholine, β-amyloid deposits, τ-protein phosphorylation, oxidative stress, and neuroinflammation are considered to play significant roles in the pathophysiology of this disease. For a long time, AD patients have been treated with acetylcholinesterase inhibitors such as donepezil (Aricept®) but with limited therapeutic success. This might be due to the complex multifactorial nature of AD, a fact that has prompted the design of new Multi-Target-Directed Ligands

  15. Multi-Target Directed Donepezil-Like Ligands for Alzheimer's Disease

    PubMed Central

    Unzeta, Mercedes; Esteban, Gerard; Bolea, Irene; Fogel, Wieslawa A.; Ramsay, Rona R.; Youdim, Moussa B. H.; Tipton, Keith F.; Marco-Contelles, José

    2016-01-01

    HIGHLIGHTS ASS234 is a MTDL compound containing a moiety from Donepezil and the propargyl group from the PF 9601N, a potent and selective MAO B inhibitor. This compound is the most advanced anti-Alzheimer agent for preclinical studies identified in our laboratory.Derived from ASS234 both multipotent donepezil-indolyl (MTDL-1) and donepezil-pyridyl hybrids (MTDL-2) were designed and evaluated as inhibitors of AChE/BuChE and both MAO isoforms. MTDL-2 showed more high affinity toward the four enzymes than MTDL-1.MTDL-3 and MTDL-4, were designed containing the N-benzylpiperidinium moiety from Donepezil, a metal- chelating 8-hydroxyquinoline group and linked to a N-propargyl core and they were pharmacologically evaluated.The presence of the cyano group in MTDL-3, enhanced binding to AChE, BuChE and MAO A. It showed antioxidant behavior and it was able to strongly complex Cu(II), Zn(II) and Fe(III).MTDL-4 showed higher affinity toward AChE, BuChE.MTDL-3 exhibited good brain penetration capacity (ADMET) and less toxicity than Donepezil. Memory deficits in scopolamine-lesioned animals were restored by MTDL-3.MTDL-3 particularly emerged as a ligand showing remarkable potential benefits for its use in AD therapy. Alzheimer's disease (AD), the most common form of adult onset dementia, is an age-related neurodegenerative disorder characterized by progressive memory loss, decline in language skills, and other cognitive impairments. Although its etiology is not completely known, several factors including deficits of acetylcholine, β-amyloid deposits, τ-protein phosphorylation, oxidative stress, and neuroinflammation are considered to play significant roles in the pathophysiology of this disease. For a long time, AD patients have been treated with acetylcholinesterase inhibitors such as donepezil (Aricept®) but with limited therapeutic success. This might be due to the complex multifactorial nature of AD, a fact that has prompted the design of new Multi-Target-Directed Ligands

  16. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. PMID:26553470

  17. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  18. Neighboring Gene Regulation by Antisense Long Non-Coding RNAs

    PubMed Central

    Villegas, Victoria E.; Zaphiropoulos, Peter G.

    2015-01-01

    Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation—pretranscriptional, transcriptional and posttranscriptional—through DNA–RNA, RNA–RNA or protein–RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm. PMID:25654223

  19. Natural antisense transcripts associated with salinity response in alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...

  20. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

    PubMed Central

    Kuraoka, Mutsuki; Lee, Joshua J.A.; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  1. Proteomic analysis of mature barley grains from C-hordein antisense lines.

    PubMed

    Schmidt, Daiana; Gaziola, Salete Aparecida; Boaretto, Luis Felipe; Azevedo, Ricardo Antunes

    2016-05-01

    Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.

  2. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    PubMed

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  3. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides.

    PubMed

    Sztainberg, Yehezkel; Chen, Hong-mei; Swann, John W; Hao, Shuang; Tang, Bin; Wu, Zhenyu; Tang, Jianrong; Wan, Ying-Wooi; Liu, Zhandong; Rigo, Frank; Zoghbi, Huda Y

    2015-12-01

    Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders. MECP2 duplication syndrome is one of the most common genomic rearrangements in males and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders, including loss of MeCP2 in Rett syndrome, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2-overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it, and have been successfully used to correct deficits in different mouse models. We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult

  4. The ICAM-1 antisense oligonucleotide ISIS-3082 prevents the development of postoperative ileus in mice.

    PubMed

    The, Frans O; de Jonge, Wouter J; Bennink, Roel J; van den Wijngaard, Rene M; Boeckxstaens, Guy E

    2005-09-01

    Intestinal manipulation (IM) during abdominal surgery triggers the influx of inflammatory cells, leading to postoperative ileus. Prevention of this local muscle inflammation, using intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1-specific antibodies, has been shown to shorten postoperative ileus. However, the therapeutic use of antibodies has considerable disadvantages. The aim of the current study was to evaluate the effect of ISIS-3082, a mouse-specific ICAM-1 antisense oligonucleotide, on postoperative ileus in mice. Mice underwent a laparotomy or a laparotomy combined with IM after treatment with ICAM-1 antibodies, 0.1-10 mg kg(-1) ISIS-3082, saline or ISIS-8997 (scrambled control antisense oligonucleotides, 1 and 3 mg kg(-1)). At 24 h after surgery, gastric emptying of a 99mTC labelled semi-liquid meal was determined using scintigraphy. Intestinal inflammation was assessed by myeloperoxidase (MPO) activity in ileal muscle whole mounts. IM significantly reduced gastric emptying compared to laparotomy. Pretreatment with ISIS-3082 (0.1-1 mg kg(-1)) as well as ICAM-1 antibodies (10 mg kg(-1)), but not ISIS-8997 or saline, improved gastric emptying in a dose-dependent manner. This effect diminished with higher doses of ISIS-3082 (3-10 mg kg(-1)). Similarly, ISIS-3082 (0.1-1 mg kg(-1)) and ICAM-1 antibodies, but not ISIS-8997 or higher doses of ISIS-3082 (3-10 mg kg(-1)), reduced manipulation-induced inflammation. Immunohistochemistry showed reduction of ICAM-1 expression with ISIS-3082 only. ISIS-3082 pretreatment prevents postoperative ileus in mice by reduction of manipulation-induced local intestinal muscle inflammation. Our data suggest that targeting ICAM-1 using antisense oligonucleotides may represent a new therapeutic approach to the prevention of postoperative ileus.

  5. Targeted Intracellular Delivery of Antisense Oligonucleotides Via Conjugation With Small Molecule Ligands

    PubMed Central

    Nakagawa, Osamu; Ming, Xin; Huang, Leaf; Juliano, Rudolph L.

    2010-01-01

    Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multi-valent conjugates of oligonucleotides with small molecule ligands. Using the sigma receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid phase DNA synthesis. We generated mono- and tri-valent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The tri-valent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the mono-valent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range. PMID:20550198

  6. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation.

    PubMed

    Park, Jehyun; Seo, Ji Yeon; Ha, Hunjoo

    2010-12-01

    Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

  7. Multi-target tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties against Alzheimer's disease.

    PubMed

    Xie, Sai-Sai; Wang, Xiaobing; Jiang, Neng; Yu, Wenying; Wang, Kelvin D G; Lan, Jin-Shuai; Li, Zhong-Rui; Kong, Ling-Yi

    2015-05-01

    A series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as multi-target agents against Alzheimer's disease. The biological assays indicated that most of compounds displayed potent inhibitory activity toward AChE and BuChE, and clearly selective inhibition for MAO-B. Among these compounds, 14c exhibited strong inhibitory activity for AChE (IC50 values of 33.63 nM for eeAChE and 16.11 nM for hAChE) and BuChE (IC50 values of 80.72 nM for eqBuChE and 112.72 nM for hBuChE), and the highest inhibitory activity against hMAO-B (IC50 value of 0.24 μM). Kinetic and molecular modeling studies revealed that 14c was a mixed-type inhibitor, binding simultaneously to catalytic, peripheral and mid-gorge sites of AChE. It was also a competitive inhibitor, which covered the substrate and entrance cavities of MAO-B. Moreover, 14c could penetrate the CNS and show low cell toxicity. Overall, these results suggested that 14c might be an excellent multi-target agent for AD treatment. PMID:25812965

  8. Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

    PubMed

    Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

    2015-04-01

    Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events. PMID:25673245

  9. Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

    PubMed

    Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

    2015-04-01

    Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

  10. Evaluation of multi-target immunogenic reagents for the detection of latent and body fluid-contaminated fingermarks.

    PubMed

    Lam, Rolanda; Hofstetter, Oliver; Lennard, Chris; Roux, Claude; Spindler, Xanthe

    2016-07-01

    Fingermark enhancement reagents capable of molecular recognition offer a highly selective and sensitive method of detection. Antibodies and aptamers provide a high degree of adaptability for visualisation, allowing for the selection of the most appropriate visualisation wavelength for a particular substrate without the need for specialist equipment or image processing. However, the major hurdle to overcome is the balance between sensitivity and selectivity. Single-target molecular recognition is highly specific, purported to have better detection limits than chemical reactions or stains, and can provide information about the donor or activity, but often results in incomplete ridge pattern development. Consequently, the development and evaluation of multi-target biomolecular reagents for fingermark enhancement was investigated, with the focus on endogenous eccrine secretions. To assess the suitability of the immunogenic reagents for potential operational use, a variety of parameters (i.e., processing time, fixing and working solution conditions) were optimised on a wide range of non-porous and semi-porous substrates. The relative performance of immunogenic reagents was compared to that of routine techniques applied to latent marks and marks in blood, semen and saliva. The incorporation of these novel reagents into routine technique sequences was also investigated. The experimental results indicated that the multi-target immunogenic reagents were not a suitable alternative to routine detection methods or sequences, but may have promise as a "last resort" method for difficult substrates or cases.

  11. In Vivo Characterization of ARN14140, a Memantine/Galantamine-Based Multi-Target Compound for Alzheimer’s Disease

    PubMed Central

    Reggiani, Angelo M.; Simoni, Elena; Caporaso, Roberta; Meunier, Johann; Keller, Emeline; Maurice, Tangui; Minarini, Anna; Rosini, Michela; Cavalli, Andrea

    2016-01-01

    Alzheimer’s disease (AD) is a chronic pathological condition that leads to neurodegeneration, loss of intellectual abilities, including cognition and memory, and ultimately to death. It is widely recognized that AD is a multifactorial disease, where different pathological cascades (mainly amyloid and tau) contribute to neural death and to the clinical outcome related to the disease. The currently available drugs for AD were developed according to the one-target, one-drug paradigm. In recent times, multi-target strategies have begun to play an increasingly central role in the discovery of more efficacious candidates for complex neurological conditions, including AD. In this study, we report on the in vivo pharmacological characterization of ARN14140, a new chemical entity, which was obtained through a multi-target structure-activity relationship campaign, and which showed a balanced inhibiting profile against the acetylcholinesterase enzyme and the NMDA receptor. Based on the initial promising biochemical data, ARN14140 is here studied in mice treated with the amyloidogenic fragment 25–35 of the amyloid-β peptide, a consolidated non-transgenic AD model. Sub-chronically treating animals with ARN14140 leads to a prevention of the cognitive impairment and of biomarker levels connected to neurodegeneration, demonstrating its neuroprotective potential as new AD agent. PMID:27609215

  12. Multi-target tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties against Alzheimer's disease.

    PubMed

    Xie, Sai-Sai; Wang, Xiaobing; Jiang, Neng; Yu, Wenying; Wang, Kelvin D G; Lan, Jin-Shuai; Li, Zhong-Rui; Kong, Ling-Yi

    2015-05-01

    A series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as multi-target agents against Alzheimer's disease. The biological assays indicated that most of compounds displayed potent inhibitory activity toward AChE and BuChE, and clearly selective inhibition for MAO-B. Among these compounds, 14c exhibited strong inhibitory activity for AChE (IC50 values of 33.63 nM for eeAChE and 16.11 nM for hAChE) and BuChE (IC50 values of 80.72 nM for eqBuChE and 112.72 nM for hBuChE), and the highest inhibitory activity against hMAO-B (IC50 value of 0.24 μM). Kinetic and molecular modeling studies revealed that 14c was a mixed-type inhibitor, binding simultaneously to catalytic, peripheral and mid-gorge sites of AChE. It was also a competitive inhibitor, which covered the substrate and entrance cavities of MAO-B. Moreover, 14c could penetrate the CNS and show low cell toxicity. Overall, these results suggested that 14c might be an excellent multi-target agent for AD treatment.

  13. In Vivo Characterization of ARN14140, a Memantine/Galantamine-Based Multi-Target Compound for Alzheimer's Disease.

    PubMed

    Reggiani, Angelo M; Simoni, Elena; Caporaso, Roberta; Meunier, Johann; Keller, Emeline; Maurice, Tangui; Minarini, Anna; Rosini, Michela; Cavalli, Andrea

    2016-01-01

    Alzheimer's disease (AD) is a chronic pathological condition that leads to neurodegeneration, loss of intellectual abilities, including cognition and memory, and ultimately to death. It is widely recognized that AD is a multifactorial disease, where different pathological cascades (mainly amyloid and tau) contribute to neural death and to the clinical outcome related to the disease. The currently available drugs for AD were developed according to the one-target, one-drug paradigm. In recent times, multi-target strategies have begun to play an increasingly central role in the discovery of more efficacious candidates for complex neurological conditions, including AD. In this study, we report on the in vivo pharmacological characterization of ARN14140, a new chemical entity, which was obtained through a multi-target structure-activity relationship campaign, and which showed a balanced inhibiting profile against the acetylcholinesterase enzyme and the NMDA receptor. Based on the initial promising biochemical data, ARN14140 is here studied in mice treated with the amyloidogenic fragment 25-35 of the amyloid-β peptide, a consolidated non-transgenic AD model. Sub-chronically treating animals with ARN14140 leads to a prevention of the cognitive impairment and of biomarker levels connected to neurodegeneration, demonstrating its neuroprotective potential as new AD agent. PMID:27609215

  14. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    PubMed

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.

  15. Multi-Target Tracking With Time-Varying Clutter Rate and Detection Profile: Application to Time-Lapse Cell Microscopy Sequences.

    PubMed

    Rezatofighi, Seyed Hamid; Gould, Stephen; Vo, Ba Tuong; Vo, Ba-Ngu; Mele, Katarina; Hartley, Richard

    2015-06-01

    Quantitative analysis of the dynamics of tiny cellular and sub-cellular structures, known as particles, in time-lapse cell microscopy sequences requires the development of a reliable multi-target tracking method capable of tracking numerous similar targets in the presence of high levels of noise, high target density, complex motion patterns and intricate interactions. In this paper, we propose a framework for tracking these structures based on the random finite set Bayesian filtering framework. We focus on challenging biological applications where image characteristics such as noise and background intensity change during the acquisition process. Under these conditions, detection methods usually fail to detect all particles and are often followed by missed detections and many spurious measurements with unknown and time-varying rates. To deal with this, we propose a bootstrap filter composed of an estimator and a tracker. The estimator adaptively estimates the required meta parameters for the tracker such as clutter rate and the detection probability of the targets, while the tracker estimates the state of the targets. Our results show that the proposed approach can outperform state-of-the-art particle trackers on both synthetic and real data in this regime. PMID:25594963

  16. Analytic performance prediction of track-to-track association with biased data in multi-sensor multi-target tracking scenarios.

    PubMed

    Tian, Wei; Wang, Yue; Shan, Xiuming; Yang, Jian

    2013-01-01

    An analytic method for predicting the performance of track-to-track association (TTTA) with biased data in multi-sensor multi-target tracking scenarios is proposed in this paper. The proposed method extends the existing results of the bias-free situation by accounting for the impact of sensor biases. Since little insight of the intrinsic relationship between scenario parameters and the performance of TTTA can be obtained by numerical simulations, the proposed analytic approach is a potential substitute for the costly Monte Carlo simulation method. Analytic expressions are developed for the global nearest neighbor (GNN) association algorithm in terms of correct association probability. The translational biases of sensors are incorporated in the expressions, which provide good insight into how the TTTA performance is affected by sensor biases, as well as other scenario parameters, including the target spatial density, the extraneous track density and the average association uncertainty error. To show the validity of the analytic predictions, we compare them with the simulation results, and the analytic predictions agree reasonably well with the simulations in a large range of normally anticipated scenario parameters. PMID:24036583

  17. Multi-Target Tracking With Time-Varying Clutter Rate and Detection Profile: Application to Time-Lapse Cell Microscopy Sequences.

    PubMed

    Rezatofighi, Seyed Hamid; Gould, Stephen; Vo, Ba Tuong; Vo, Ba-Ngu; Mele, Katarina; Hartley, Richard

    2015-06-01

    Quantitative analysis of the dynamics of tiny cellular and sub-cellular structures, known as particles, in time-lapse cell microscopy sequences requires the development of a reliable multi-target tracking method capable of tracking numerous similar targets in the presence of high levels of noise, high target density, complex motion patterns and intricate interactions. In this paper, we propose a framework for tracking these structures based on the random finite set Bayesian filtering framework. We focus on challenging biological applications where image characteristics such as noise and background intensity change during the acquisition process. Under these conditions, detection methods usually fail to detect all particles and are often followed by missed detections and many spurious measurements with unknown and time-varying rates. To deal with this, we propose a bootstrap filter composed of an estimator and a tracker. The estimator adaptively estimates the required meta parameters for the tracker such as clutter rate and the detection probability of the targets, while the tracker estimates the state of the targets. Our results show that the proposed approach can outperform state-of-the-art particle trackers on both synthetic and real data in this regime.

  18. Analytic performance prediction of track-to-track association with biased data in multi-sensor multi-target tracking scenarios.

    PubMed

    Tian, Wei; Wang, Yue; Shan, Xiuming; Yang, Jian

    2013-09-12

    An analytic method for predicting the performance of track-to-track association (TTTA) with biased data in multi-sensor multi-target tracking scenarios is proposed in this paper. The proposed method extends the existing results of the bias-free situation by accounting for the impact of sensor biases. Since little insight of the intrinsic relationship between scenario parameters and the performance of TTTA can be obtained by numerical simulations, the proposed analytic approach is a potential substitute for the costly Monte Carlo simulation method. Analytic expressions are developed for the global nearest neighbor (GNN) association algorithm in terms of correct association probability. The translational biases of sensors are incorporated in the expressions, which provide good insight into how the TTTA performance is affected by sensor biases, as well as other scenario parameters, including the target spatial density, the extraneous track density and the average association uncertainty error. To show the validity of the analytic predictions, we compare them with the simulation results, and the analytic predictions agree reasonably well with the simulations in a large range of normally anticipated scenario parameters.

  19. Dovitinib (TKI258), a multi-target angiokinase inhibitor, is effective regardless of KRAS or BRAF mutation status in colorectal cancer

    PubMed Central

    Lee, Choong-Kun; Lee, Myung Eun; Lee, Won Suk; Kim, Jeong Min; Park, Kyu Hyun; Kim, Tae Soo; Lee, Kang Young; Ahn, Joong Bae; Chung, Hyun Cheol; Rha, Sun Young

    2015-01-01

    Introduction: We aimed to determine whether KRAS and BRAF mutant colorectal cancer (CRC) cells exhibit distinct sensitivities to the multi-target angiokinase inhibitor, TKI258 (dovitinib). Materials and methods: We screened 10 CRC cell lines by using receptor tyrosine kinase (RTK) array to identify activated RTKs. MTT assays, anchorage-independent colony-formation assays, and immunoblotting assays were performed to evaluate the in vitro anti-tumor effects of TKI258. In vivo efficacy study followed by pharmacodynamic evaluation was done. Results: Fibroblast Growth Factor Receptor 1 (FGFR1) and FGFR3 were among the most highly activated RTKs in CRC cell lines. In in vitro assays, the BRAF mutant HT-29 cells were more resistant to the TKI258 than the KRAS mutant LoVo cells. However, in xenograft assays, TKI258 equally delayed the growth of tumors induced by both cell lines. TUNEL assays showed that the apoptotic index was unchanged following TKI258 treatment, but staining for Ki-67 and CD31 was substantially reduced in both xenografts, implying an anti-angiogenic effect of the drug. TKI258 treatment was effective in delaying CRC tumor growth in vivo regardless of the KRAS and BRAF mutation status. Conclusions: Our results identify FGFRs as potential targets in CRC treatment and suggest that combined targeting of multiple RTKs with TKI258 might serve as a novel approach to improve outcome of patients with CRC. PMID:25628921

  20. Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi

    PubMed Central

    Fowler, Daniel K.; Williams, Carly; Gerritsen, Alida T.; Washbourne, Philip

    2016-01-01

    Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired. PMID:26582923

  1. Prolonged-acting, multi-targeting gallium nanoparticles potently inhibit growth of both HIV and mycobacteria in co-infected human macrophages.

    PubMed

    Narayanasamy, Prabagaran; Switzer, Barbara L; Britigan, Bradley E

    2015-03-06

    Human immunodeficiency virus (HIV) infection and Mycobacterium tuberculosis (TB) are responsible for two of the major global human infectious diseases that result in significant morbidity, mortality and socioeconomic impact. Furthermore, severity and disease prevention of both infections is enhanced by co-infection. Parallel limitations also exist in access to effective drug therapy and the emergence of resistance. Furthermore, drug-drug interactions have proven problematic during treatment of co-incident HIV and TB infections. Thus, improvements in drug access and simplified treatment regimens are needed immediately. One of the key host cells infected by both HIV and TB is the mononuclear phagocyte (MP; monocyte, macrophage and dendritic cell). Therefore, we hypothesized that one way this can be achieved is through drug-targeting by a nanoformulated drug that ideally would be active against both HIV and TB. Accordingly, we validated macrophage targeted long acting (sustained drug release) gallium (Ga) nanoformulation against HIV-mycobacterium co-infection. The multi-targeted Ga nanoparticle agent inhibited growth of both HIV and TB in the macrophage. The Ga nanoparticles reduced the growth of mycobacterium and HIV for up to 15 days following single drug loading. These results provide a potential new approach to treat HIV-TB co-infection that could eventually lead to improved clinical outcomes.

  2. Prolonged-acting, Multi-targeting Gallium Nanoparticles Potently Inhibit Growth of Both HIV and Mycobacteria in Co-Infected Human Macrophages

    PubMed Central

    Narayanasamy, Prabagaran; Switzer, Barbara L.; Britigan, Bradley E.

    2015-01-01

    Human immunodeficiency virus (HIV) infection and Mycobacterium tuberculosis (TB) are responsible for two of the major global human infectious diseases that result in significant morbidity, mortality and socioeconomic impact. Furthermore, severity and disease prevention of both infections is enhanced by co-infection. Parallel limitations also exist in access to effective drug therapy and the emergence of resistance. Furthermore, drug-drug interactions have proven problematic during treatment of co-incident HIV and TB infections. Thus, improvements in drug access and simplified treatment regimens are needed immediately. One of the key host cells infected by both HIV and TB is the mononuclear phagocyte (MP; monocyte, macrophage and dendritic cell). Therefore, we hypothesized that one way this can be achieved is through drug-targeting by a nanoformulated drug that ideally would be active against both HIV and TB. Accordingly, we validated macrophage targeted long acting (sustained drug release) gallium (Ga) nanoformulation against HIV-mycobacterium co-infection. The multi-targeted Ga nanoparticle agent inhibited growth of both HIV and TB in the macrophage. The Ga nanoparticles reduced the growth of mycobacterium and HIV for up to 15 days following single drug loading. These results provide a potential new approach to treat HIV-TB co-infection that could eventually lead to improved clinical outcomes. PMID:25744727

  3. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  4. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    PubMed Central

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns—multisystemic, hepatic, or in central nervous system (CNS)—and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  5. Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance.

    PubMed

    Iwaki, T; Iwaki, A; Tateishi, J; Goldman, J E

    1994-06-01

    The phenotypic effects of selectively altering the levels of alpha B-crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B-crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization. PMID:8207065

  6. Antisense suppression of an acid invertase gene (MAI1) in muskmelon alters plant growth and fruit development.

    PubMed

    Yu, Xiyan; Wang, Xiufeng; Zhang, Wenqian; Qian, Tingting; Tang, Guimin; Guo, Yankui; Zheng, Chengchao

    2008-01-01

    To unravel the roles of soluble acid invertase in muskmelon (Cucumis melo L.), its activity in transgenic muskmelon plants was reduced by an antisense approach. For this purpose, a 1038 bp cDNA fragment of muskmelon soluble acid invertase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the stems were obviously thinner. Transmission electron microscopy revealed that degradation of the chloroplast membrane occurred in transgenic leaves and the number of grana in the chloroplast was significantly reduced, suggesting that the slow growth and weaker phenotype of the transgenic plants may be due to damage to the chloroplast ultrastructure, which in turn resulted in a decrease in net photosynthetic rate. The sucrose concentration increased and levels of acid invertase decreased in transgenic fruit, and the fruit size was 60% smaller than that of the control. In addition, transgenic fruit reached full-slip at 25 d after pollination (DAP), approximately 5 d before the control fruit (full-slip at 30 DAP), and this accelerated maturity correlated with a dramatic elevation of ethylene production at the later stages of fruit development. Together, these results suggest that soluble acid invertase not only plays an important role during muskmelon plant and fruit development but also controls the sucrose content in muskmelon fruit.

  7. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    PubMed

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  8. Bacterial antisense RNAs are mainly the product of transcriptional noise

    PubMed Central

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  9. Bacterial antisense RNAs are mainly the product of transcriptional noise.

    PubMed

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

    2016-03-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  10. Multi target neuroprotective and neurorestorative anti-Parkinson and anti-Alzheimer drugs ladostigil and m30 derived from rasagiline.

    PubMed

    Youdim, Moussa B H

    2013-03-01

    Present anti-PD and -AD drugs have limited symptomatic activity and devoid of neuroprotective and neurorestorative property that is needed for disease modifying action. The complex pathology of PD and AD led us to develop several multi-target neuroprotective and neurorestorative drugs with several CNS targets with the ability for possible disease modifying activity. Employing the pharmacophore of our anti-parkinson drug rasagiline (Azilect, N-propagrgyl-1-R-aminoindan), we have developed a series of novel multi-functional neuroprotective drugs (A) [TV-3326 (N-propargyl-3R-aminoindan-5yl)-ethyl methylcarbamate)], with both cholinesterase-butyrylesterase and brain selective monoamine-oxidase (MAO) A/B inhibitory activities and (B) the iron chelator-radical scavenging-brain selective monoamine oxidase (MAO) A/B inhibitor and M30 possessing the neuroprotective and neurorescuing propargyl moiety of rasagiline, as potential treatment of AD, DLB and PD with dementia. Another series of multi-target drugs (M30, HLA-20 series) which are brain permeable iron chelators and potent selective brain MAO inhibitors were also developed. These series of drugs have the ability of regulating and processing amyloid precursor protein (APP) since APP and alpha-synuclein are metaloproteins (iron-regulated proteins), with an iron responsive element 5"UTR mRNA similar to transferring and ferritin. Ladostigil inhibits brain acetyl and butyrylcholinesterase in rats after oral doses. After chronic but not acute treatment, it inhibits MAO-A and -B in the brain. Ladostigil acts like an anti-depressant in the forced swim test in rats, indicating a potential for anti-depressant activity. Ladostigil prevents the destruction of nigrostriatal neurons induced by infusion of neurotoxin MPTP in mice. The propargylamine moiety of ladostigil confers neuroprotective activity against cytotoxicity induced by ischemia and peroxynitrite in cultured neuronal cells. The multi-target iron chelator M30 has all the

  11. Antisense oligonucleotides: is the glass half full or half empty?

    PubMed

    Bennett, C F

    1998-01-01

    Antisense oligonucleotides are widely used as tools to explore the pharmacological effects of inhibiting expression of a selected gene product. In addition, they are being investigated as therapeutic agents for the treatment of viral infections, cancers, and inflammatory disorders. Proof that the pharmacological effects produced by the oligonucleotides are attributable to an antisense mechanism of action requires careful experimentation. Central to this problem is the finding that oligonucleotides are capable of interacting with and modulating function of specific proteins in both a sequence-independent and -dependent manner. Despite these undesired interactions, it has been possible to demonstrate that oligonucleotides are capable of binding to a specific RNA in cultured cells, or within tissues, resulting in selective reduction of the targeted gene product and pharmacological activity. In general, these oligonucleotides were identified after a selection process in which multiple oligonucleotides targeting different regions on the RNA were evaluated for direct inhibition of targeted gene product, resulting in the identification of a potent and selective oligonucleotide. Similar to other drug-receptor interactions, selection of the most potent inhibitor results in an increase in the signal-to-noise ratio, yielding increased confidence that activity observed is the result of a desired effect of the inhibitor. With careful selection, proper controls, and careful dose-response curves it is possible to utilize antisense oligonucleotides as effective research tools and potentially as therapeutic agents. PMID:9413924

  12. Multi-targeted inhibition of tumor growth and lung metastasis by redox-sensitive shell crosslinked micelles loading disulfiram

    NASA Astrophysics Data System (ADS)

    Duan, Xiaopin; Xiao, Jisheng; Yin, Qi; Zhang, Zhiwen; Yu, Haijun; Mao, Shirui; Li, Yaping

    2014-03-01

    Metastasis, the main cause of cancer related deaths, remains the greatest challenge in cancer treatment. Disulfiram (DSF), which has multi-targeted anti-tumor activity, was encapsulated into redox-sensitive shell crosslinked micelles to achieve intracellular targeted delivery and finally inhibit tumor growth and metastasis. The crosslinked micelles demonstrated good stability in circulation and specifically released DSF under a reductive environment that mimicked the intracellular conditions of tumor cells. As a result, the DSF-loaded redox-sensitive shell crosslinked micelles (DCMs) dramatically inhibited cell proliferation, induced cell apoptosis and suppressed cell invasion, as well as impairing tube formation of HMEC-1 cells. In addition, the DCMs could accumulate in tumor tissue and stay there for a long time, thereby causing significant inhibition of 4T1 tumor growth and marked prevention in lung metastasis of 4T1 tumors. These results suggested that DCMs could be a promising delivery system in inhibiting the growth and metastasis of breast cancer.

  13. Simultaneous SPECT imaging of multi-targets to assist in identifying hepatic lesions

    PubMed Central

    Guo, Zhide; Gao, Mengna; Zhang, Deliang; Li, Yesen; Song, Manli; Zhuang, Rongqiang; Su, Xinhui; Chen, Guibing; Liu, Ting; Liu, Pingguo; Wu, Hua; Du, Jin; Zhang, Xianzhong

    2016-01-01

    Molecular imaging technique is an attractive tool to detect liver disease at early stage. This study aims to develop a simultaneous dual-isotope single photon emission computed tomography (SPECT)/CT imaging method to assist diagnosis of hepatic tumor and liver fibrosis. Animal models of liver fibrosis and orthotopic human hepatocellular carcinoma (HCC) were established. The tracers of 131I-NGA and 99mTc-3P-RGD2 were selected to target asialoglycoprotein receptor (ASGPR) on the hepatocytes and integrin αvβ3 receptor in tumor or fibrotic liver, respectively. SPECT imaging and biodistribution study were carried out to verify the feasibility and superiority. As expected, 99mTc-3P-RGD2 had the ability to evaluate liver fibrosis and detect tumor lesions. 131I-NGA showed that it was effective in assessing the anatomy and function of the liver. In synchronized dual-isotope SPECT/CT imaging, clear fusion images can be got within 30 minutes for diagnosing liver fibrosis and liver cancer. This new developed imaging approach enables the acquisition of different physiological information for diagnosing liver fibrosis, liver cancer and evaluating residual functional liver volume simultaneously. So synchronized dual-isotope SPECT/CT imaging with 99mTc-3P-RGD2 and 131I-NGA is an effective approach to detect liver disease, especially liver fibrosis and liver cancer. PMID:27377130

  14. Online Multi-Target Tracking With Unified Handling of Complex Scenarios.

    PubMed

    Jiang, Huaizu; Wang, Jinjun; Gong, Yihong; Rong, Na; Chai, Zhenhua; Zheng, Nanning

    2015-11-01

    Complex scenarios, including miss detections, occlusions, false detections, and trajectory terminations, make the data association challenging. In this paper, we propose an online tracking-by-detection method to track multiple targets with unified handling of aforementioned complex scenarios, where current detection responses are linked to the previous trajectories. We introduce a dummy node to each trajectory to allow it to temporally disappear. If a trajectory fails to find its matching detection, it will be linked to its corresponding dummy node until the emergence of its matching detection. Source nodes are also incorporated to account for the entrance of new targets. The standard Hungarian algorithm, extended by the dummy nodes, can be exploited to solve the online data association implicitly in a global manner, although it is formulated between two consecutive frames. Moreover, as dummy nodes tend to accumulate in a fake or disappeared trajectory while they only occasionally appear in a real trajectory, we can deal with false detections and trajectory terminations by simply checking the number of consecutive dummy nodes. Our approach works on a single, uncalibrated camera, and requires neither scene prior knowledge nor explicit occlusion reasoning, running at 132 frames/s on the PETS09-S2L1 benchmark sequence. The experimental results validate the effectiveness of the dummy nodes in complex scenarios and show that our proposed approach is robust against false detections and miss detections. Quantitative comparisons with other methods on five benchmark sequences demonstrate that we can achieve comparable results with the most existing offline methods and better results than other online algorithms. PMID:26087489

  15. Behavior-based cooperative robotics applied to multi-target observation

    SciTech Connect

    Parker, L.E.

    1996-12-31

    An important issue that arises in the automation of many security, surveillance, and reconnaissance tasks is that of monitoring (or observing) the movements of targets navigating in a bounded area of interest. A key research issue in these problems is that of sensor placement - determining where sensors should be located to maintain the targets in view. In complex applications involving limited-range sensors, the use of multiple sensors dynamically moving over time is required. In this paper, the author investigates the use of a cooperative team of autonomous sensor-based robots for the observation of multiple moving targets. The author focuses primarily on developing the distributed control strategies that allow the robot team to attempt to minimize the total time in which targets escape observation by some robot team member in the area of interest. The initial efforts on this problem address the aspects of distributed control in homogeneous robot teams with equivalent sensing and movement capabilities working in an uncluttered, bounded area. This paper first formalizes the problem, discusses related work, and then shows that this problem is NP-hard. The author then presents a distributed approximate approach to solving this problem that combines low-level multi-robot control with higher-level control. The low-level control is described in terms of force fields emanating from the targets and the robots. The higher level control is presented in the ALLIANCE formalism, which provides mechanisms for fault tolerant cooperative control, and allows robot team members to adjust their low-level actions based upon the actions of their teammates. The author then presents the results of the ongoing implementation of this approach, both in simulation and on physical robots. To the authors knowledge, this is the first paper addressing this research problem that has been implemented on physical robot teams.

  16. Online Multi-Target Tracking With Unified Handling of Complex Scenarios.

    PubMed

    Jiang, Huaizu; Wang, Jinjun; Gong, Yihong; Rong, Na; Chai, Zhenhua; Zheng, Nanning

    2015-11-01

    Complex scenarios, including miss detections, occlusions, false detections, and trajectory terminations, make the data association challenging. In this paper, we propose an online tracking-by-detection method to track multiple targets with unified handling of aforementioned complex scenarios, where current detection responses are linked to the previous trajectories. We introduce a dummy node to each trajectory to allow it to temporally disappear. If a trajectory fails to find its matching detection, it will be linked to its corresponding dummy node until the emergence of its matching detection. Source nodes are also incorporated to account for the entrance of new targets. The standard Hungarian algorithm, extended by the dummy nodes, can be exploited to solve the online data association implicitly in a global manner, although it is formulated between two consecutive frames. Moreover, as dummy nodes tend to accumulate in a fake or disappeared trajectory while they only occasionally appear in a real trajectory, we can deal with false detections and trajectory terminations by simply checking the number of consecutive dummy nodes. Our approach works on a single, uncalibrated camera, and requires neither scene prior knowledge nor explicit occlusion reasoning, running at 132 frames/s on the PETS09-S2L1 benchmark sequence. The experimental results validate the effectiveness of the dummy nodes in complex scenarios and show that our proposed approach is robust against false detections and miss detections. Quantitative comparisons with other methods on five benchmark sequences demonstrate that we can achieve comparable results with the most existing offline methods and better results than other online algorithms.

  17. Antisense-Based Progerin Downregulation in HGPS-Like Patients’ Cells

    PubMed Central

    Harhouri, Karim; Navarro, Claire; Baquerre, Camille; Da Silva, Nathalie; Bartoli, Catherine; Casey, Frank; Mawuse, Guedenon Koffi; Doubaj, Yassamine; Lévy, Nicolas; De Sandre-Giovannoli, Annachiara

    2016-01-01

    Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named “HGPS-like” patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90) at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON) prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90) in HGPS-like patients’ cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A), was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients. PMID:27409638

  18. Multi-target detection and estimation with the use of massive independent, identical sensors

    NASA Astrophysics Data System (ADS)

    Li, Tiancheng; Corchado, Juan M.; Bajo, Javier; Chen, Genshe

    2015-05-01

    This paper investigates the problem of using a large number of independent, identical sensors jointly for multi-object detection and estimation (MODE), namely massive sensor MODE. This is significantly different to the general target tracking using few sensors. The massive sensor data allows very accurate estimation in theory (but may instead go conversely in fact) but will also cause a heavy computational burden for the traditional filter-based tracker. Instead, we propose a clustering method to fuse massive sensor data in the same state space, which is shown to be able to filter clutter and to estimate states of the targets without the use of any traditional filter. This non-Bayesian solution as referred to massive sensor observation-only (O2) inference needs neither to assume the target/clutter model nor to know the system noises. Therefore it can handle challenging scenarios with few prior information and do so very fast computationally. Simulations with the use of massive homogeneous (independent identical distributed) sensors have demonstrated the validity and superiority of the proposed approach.

  19. Multi-targeted mechanisms underlying the endothelial protective effects of the diabetic-safe sweetener erythritol.

    PubMed

    Boesten, Daniëlle M P H J; Berger, Alvin; de Cock, Peter; Dong, Hua; Hammock, Bruce D; den Hartog, Gertjan J M; Bast, Aalt

    2013-01-01

    Diabetes is characterized by hyperglycemia and development of vascular pathology. Endothelial cell dysfunction is a starting point for pathogenesis of vascular complications in diabetes. We previously showed the polyol erythritol to be a hydroxyl radical scavenger preventing endothelial cell dysfunction onset in diabetic rats. To unravel mechanisms, other than scavenging of radicals, by which erythritol mediates this protective effect, we evaluated effects of erythritol in endothelial cells exposed to normal (7 mM) and high glucose (30 mM) or diabetic stressors (e.g. SIN-1) using targeted and transcriptomic approaches. This study demonstrates that erythritol (i.e. under non-diabetic conditions) has minimal effects on endothelial cells. However, under hyperglycemic conditions erythritol protected endothelial cells against cell death induced by diabetic stressors (i.e. high glucose and peroxynitrite). Also a number of harmful effects caused by high glucose, e.g. increased nitric oxide release, are reversed. Additionally, total transcriptome analysis indicated that biological processes which are differentially regulated due to high glucose are corrected by erythritol. We conclude that erythritol protects endothelial cells during high glucose conditions via effects on multiple targets. Overall, these data indicate a therapeutically important endothelial protective effect of erythritol under hyperglycemic conditions.

  20. Molecular mechanisms of the neuroprotective/neurorescue action of multi-target green tea polyphenols.

    PubMed

    Mandel, Silvia A; Weinreb, Orly; Amit, Tamar; Youdim, Moussa B H

    2012-01-01

    Mounting evidence suggests that lifestyle factors, especially nutrition are essential factor for healthy ageing. However, as a result of the increase in life expectance, neurodegenerative diseases like Alzheimer's and Parkinson's (AD and PD, respectively) are becoming an increasing burden, as aging is their main risk factor. Brain aging and neurodegenerative diseases of the elderly are characterized by oxidative damage, dysregulation of redox metals homeostasis and inflammation. Thus, it is not surprising that a large amount of drugs/agents in therapeutic use for these conditions are antioxidants/metal complexing, bioenergetic and anti-inflammatory agents. Natural plant polyphenols (flavonoids and non-flavonoids) are the most abundant antioxidants in the diet and as such, are ideal nutraceuticals for neutralizing stress-induced free radicals and inflammation. Human epidemiological and new animal data suggest that green and black flavonoids named catechins, may help protecting the aging brain and reduce the incidence of dementia, AD and PD. This review will present salient features of the beneficial multi-pharmacological actions of black and green tea polyphenols in aging and neurodegeneration, and speculate on their potential in drug combination to target distinct pathologies as a therapeutic disease modification approach.

  1. A modeling study for structure features of β-N-acetyl-D-hexosaminidase from Ostrinia furnacalis and its novel inhibitor allosamidin: species selectivity and multi-target characteristics.

    PubMed

    Wang, Yanli; Liu, Tian; Yang, Qing; Li, Zhong; Qian, Xuhong

    2012-04-01

    Insect β-N-acetyl-D-hexosaminidase, a chitin degrading enzyme, is physiologically important during the unique life cycle of the insect. OfHex1, a β-N-acetyl-D-hexosaminidase from the insect, Ostrinia furna, which was obtained by our laboratory (Gen Bank No.: ABI81756.1), was studied by molecular modeling as well as by molecular docking with its inhibitor, allosamidin. 3D model of OfHex1 was built through the ligand-supported homology modeling approach. The binding modes of its substrate and inhibitor were proposed through docking and cluster analysis. The pocket's size and shape of OfHex1 differ from that of human β-N-acetyl-D-hexosaminidase, which determined that allosamidin can selectively inhibit OfHex1 instead of human β-N-acetyl-D-hexosaminidase. Moreover, the multi-target characteristics of allosamidin that inhibit enzymes from different families, OfHex1 (EC 3.2.1.52; GH20) and chitinase (EC 3.2.1.14; GH18), were compared. The common -1/+1 sugar-binding site of chitinase and OfHex1, and the -2/-3 sugar-binding site in chitinase contribute to the binding of allosamidin. This work, at molecular level, proved that OfHex1 could be a potential species-specific target for novel green pesticide design and also provide the possibility to develop allosamidin or its derivatives as a new type of insecticide to 'hit two birds with one stone', which maybe become a novel strategy in pest control. PMID:22177554

  2. Synthesis and evaluation of multi-target-directed ligands for the treatment of Alzheimer's disease based on the fusion of donepezil and melatonin.

    PubMed

    Wang, Jin; Wang, Zhi-Min; Li, Xue-Mei; Li, Fan; Wu, Jia-Jia; Kong, Ling-Yi; Wang, Xiao-Bing

    2016-09-15

    A novel series of compounds obtained by fusing the acetylcholinesterase (AChE) inhibitor donepezil and the antioxidant melatonin were designed as multi-target-directed ligands for the treatment of Alzheimer's disease (AD). In vitro assay indicated that most of the target compounds exhibited a significant ability to inhibit acetylcholinesterase (eeAChE and hAChE), butyrylcholinesterase (eqBuChE and hBuChE), and β-amyloid (Aβ) aggregation, and to act as potential antioxidants and biometal chelators. Especially, 4u displayed a good inhibition of AChE (IC50 value of 193nM for eeAChE and 273nM for hAChE), strong inhibition of BuChE (IC50 value of 73nM for eqBuChE and 56nM for hBuChE), moderate inhibition of Aβ aggregation (56.3% at 20μM) and good antioxidant activity (3.28trolox equivalent by ORAC assay). Molecular modeling studies in combination with kinetic analysis revealed that 4u was a mixed-type inhibitor, binding simultaneously to catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, 4u could chelate metal ions, reduce PC12 cells death induced by oxidative stress and penetrate the blood-brain barrier (BBB). Taken together, these results strongly indicated the hybridization approach is an efficient strategy to identify novel scaffolds with desired bioactivities, and further optimization of 4u may be helpful to develop more potent lead compound for AD treatment. PMID:27460699

  3. In silico search for multi-target therapies for osteoarthritis based on 10 common Huoxue Huayu herbs and potential applications to other diseases.

    PubMed

    Zheng, Chun-Song; Zhuang, Zhi-Qiang; Xu, Xiao-Jie; Ye, Jin-Xia; Ye, Hong-Zhi; Li, Xi-Hai; Wu, Guang-Wen; Xu, Hui-Feng; Liu, Xian-Xiang

    2014-03-01

    Huoxue Huayu (HXHY) has been widely used in traditional Chinese medicine (TCM) as a key therapeutic principle for osteoarthritis (OA), and related herbs have been widely prescribed to treat OA in the clinic. The aims of the present study were to explore a multi-target therapy for OA using 10 common HXHY herbs and to investigate their potential applications for treatment of other diseases. A novel computational simulation approach that integrates chemical structure, ligand clusters, chemical space and drug‑likeness evaluations, as well as docking and network analysis, was used to investigate the properties and effects of the herbs. The compounds contained in the studied HXHY herbs were divided into 10 clusters. Comparison of the chemical properties of these compounds to those of other compounds described in the DrugBank database indicated that the properties of the former are more diverse than those of the latter and that most of the HXHY-derived compounds do not violate the 'Lipinski's rule of five'. Docking analysis allowed for the identification of 39 potential bioactive compounds from HXHY herbs and 11 potential targets for these compounds. The identified targets were closely associated with 49 diseases, including neoplasms, musculoskeletal, nervous system and cardiovascular diseases. Ligand‑target (L‑T) and ligand‑target‑disease (L‑T‑D) networks were constructed in order to further elucidate the pharmacological effects of the herbs. Our findings suggest that a number of compounds from HXHY herbs are promising candidates for mult‑target therapeutic application in OA and may exert diverse pharmacological effects, affecting additional diseases besides OA.

  4. How to escape the cancer attractor: rationale and limitations of multi-target drugs.

    PubMed

    Huang, Sui; Kauffman, Stuart

    2013-08-01

    The increasingly evident limitations of target-selective cancer therapy has stimulated a flurry of ideas for overcoming the development of resistance and recurrence - the near universal reason for therapy failure from which target-selective drugs are not exempt. A widely proposed approach to conquer therapy resistance is to depart from the myopic focus on individual causal pathways and instead target multiple nodes in the cancer cell's gene regulatory network. However, most ideas rely on a simplistic conceptualization of networks: utilizing solely their topology and treating it as a display of causal interactions, while ignoring the integrated dynamics in state space. Here, we review the more encompassing formal framework of global network dynamics in which cancer cells, like normal cell types, are high-dimensional attractor states. Then therapy is represented by the network perturbation that will promote the exit from such cancer attractors and reentering a normal attractor. We show in this qualitative and accessible discussion how the idea of a quasi-potential landscape and the theory of least-action-path offer a new formal understanding for computing the set of network nodes (molecular targets) that need to be targeted in concert in order to exit the cancer attractor. But targeting cancer cells based on the network configuration of an "average" cancer cell, however precise, may not suffice to eradicate all tumor cells because of the dynamic non-genetic heterogeneity of cancer cell populations that makes them moving targets and drives the replenishment of the cancer attractor with surviving, non-responsive cells from neighboring abnormal attractors.

  5. Pseudogenes as an alternative source of natural antisense transcripts

    PubMed Central

    2010-01-01

    Background Naturally occurring antisense transcripts (NATs) are non-coding RNAs that may regulate the activity of sense transcripts to which they bind because of complementarity. NATs that are not located in the gene they regulate (trans-NATs) have better chances to evolve than cis-NATs, which is evident when the sense strand of the cis-NAT is part of a protein coding gene. However, the generation of a trans-NAT requires the formation of a relatively large region of complementarity to the gene it regulates. Results Pseudogene formation may be one evolutionary mechanism that generates trans-NATs to the parental gene. For example, this could occur if the parental gene is regulated by a cis-NAT that is copied as a trans-NAT in the pseudogene. To support this we identified human pseudogenes with a trans-NAT to the parental gene in their antisense strand by analysis of the database of expressed sequence tags (ESTs). We found that the mutations that appeared in these trans-NATs after the pseudogene formation do not show the flat distribution that would be expected in a non functional transcript. Instead, we found higher similarity to the parental gene in a region nearby the 3' end of the trans-NATs. Conclusions Our results do not imply a functional relation of the trans-NAT arising from pseudogenes over their respective parental genes but add evidence for it and stress the importance of duplication mechanisms of genetic material in the generation of non-coding RNAs. We also provide a plausible explanation for the large transcripts that can be found in the antisense strand of some pseudogenes. PMID:21047404

  6. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    ERIC Educational Resources Information Center

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  7. Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: Role of norepinephrine and acetylcholine

    PubMed Central

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 μL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the β-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala. PMID:18772255

  8. Multi-Target-Directed Ligands and other Therapeutic Strategies in the Search of a Real Solution for Alzheimer's Disease

    PubMed Central

    Agis-Torres, Angel; Sölhuber, Monica; Fernandez, Maria; Sanchez-Montero, J.M.

    2014-01-01

    The lack of an adequate therapy for Alzheimer's Disease (AD) contributes greatly to the continuous growing amount of papers and reviews, reflecting the important efforts made by scientists in this field. It is well known that AD is the most common cause of dementia, and up-to-date there is no prevention therapy and no cure for the disease, which contrasts with the enormous efforts put on the task. On the other hand many aspects of AD are currently debated or even unknown. This review offers a view of the current state of knowledge about AD which includes more relevant findings and processes that take part in the disease; it also shows more relevant past, present and future research on therapeutic drugs taking into account the new paradigm “Multi-Target-Directed Ligands” (MTDLs). In our opinion, this paradigm will lead from now on the research toward the discovery of better therapeutic solutions, not only in the case of AD but also in other complex diseases. This review highlights the strategies followed by now, and focuses other emerging targets that should be taken into account for the future development of new MTDLs. Thus, the path followed in this review goes from the pathology and the processes involved in AD to the strategies to consider in on-going and future researches. PMID:24533013

  9. Multi-Target Joint Detection and Estimation Error Bound for the Sensor with Clutter and Missed Detection.

    PubMed

    Lian, Feng; Zhang, Guang-Hua; Duan, Zhan-Sheng; Han, Chong-Zhao

    2016-01-01

    The error bound is a typical measure of the limiting performance of all filters for the given sensor measurement setting. This is of practical importance in guiding the design and management of sensors to improve target tracking performance. Within the random finite set (RFS) framework, an error bound for joint detection and estimation (JDE) of multiple targets using a single sensor with clutter and missed detection is developed by using multi-Bernoulli or Poisson approximation to multi-target Bayes recursion. Here, JDE refers to jointly estimating the number and states of targets from a sequence of sensor measurements. In order to obtain the results of this paper, all detectors and estimators are restricted to maximum a posteriori (MAP) detectors and unbiased estimators, and the second-order optimal sub-pattern assignment (OSPA) distance is used to measure the error metric between the true and estimated state sets. The simulation results show that clutter density and detection probability have significant impact on the error bound, and the effectiveness of the proposed bound is verified by indicating the performance limitations of the single-sensor probability hypothesis density (PHD) and cardinalized PHD (CPHD) filters for various clutter densities and detection probabilities. PMID:26828499

  10. A multi-target real-time PCR assay for rapid identification of meningitis-associated microorganisms.

    PubMed

    Favaro, Marco; Savini, Vincenzo; Favalli, Cartesio; Fontana, Carla

    2013-01-01

    A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient's cerebrospinal fluid (CSF) is currently considered the "gold standard" for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.

  11. Multi-Target Joint Detection and Estimation Error Bound for the Sensor with Clutter and Missed Detection.

    PubMed

    Lian, Feng; Zhang, Guang-Hua; Duan, Zhan-Sheng; Han, Chong-Zhao

    2016-01-28

    The error bound is a typical measure of the limiting performance of all filters for the given sensor measurement setting. This is of practical importance in guiding the design and management of sensors to improve target tracking performance. Within the random finite set (RFS) framework, an error bound for joint detection and estimation (JDE) of multiple targets using a single sensor with clutter and missed detection is developed by using multi-Bernoulli or Poisson approximation to multi-target Bayes recursion. Here, JDE refers to jointly estimating the number and states of targets from a sequence of sensor measurements. In order to obtain the results of this paper, all detectors and estimators are restricted to maximum a posteriori (MAP) detectors and unbiased estimators, and the second-order optimal sub-pattern assignment (OSPA) distance is used to measure the error metric between the true and estimated state sets. The simulation results show that clutter density and detection probability have significant impact on the error bound, and the effectiveness of the proposed bound is verified by indicating the performance limitations of the single-sensor probability hypothesis density (PHD) and cardinalized PHD (CPHD) filters for various clutter densities and detection probabilities.

  12. TimeLapseAnalyzer: multi-target analysis for live-cell imaging and time-lapse microscopy.

    PubMed

    Huth, Johannes; Buchholz, Malte; Kraus, Johann M; Mølhave, Kristian; Gradinaru, Cristian; v Wichert, Götz; Gress, Thomas M; Neumann, Heiko; Kestler, Hans A

    2011-11-01

    The direct observation of cells over time using time-lapse microscopy can provide deep insights into many important biological processes. Reliable analyses of motility, proliferation, invasive potential or mortality of cells are essential to many studies involving live cell imaging and can aid in biomarker discovery and diagnostic decisions. Given the vast amount of image- and time-series data produced by modern microscopes, automated analysis is a key feature to capitalize the potential of time-lapse imaging devices. To provide fast and reproducible analyses of multiple aspects of cell behaviour, we developed TimeLapseAnalyzer. Apart from general purpose image enhancements and segmentation procedures, this extensible, self-contained, modular cross-platform package provides dedicated modalities for fast and reliable analysis of multi-target cell tracking, scratch wound healing analysis, cell counting and tube formation analysis in high throughput screening of live-cell experiments. TimeLapseAnalyzer is freely available (MATLAB, Open Source) at http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/tla.

  13. Multi-Target Joint Detection and Estimation Error Bound for the Sensor with Clutter and Missed Detection

    PubMed Central

    Lian, Feng; Zhang, Guang-Hua; Duan, Zhan-Sheng; Han, Chong-Zhao

    2016-01-01

    The error bound is a typical measure of the limiting performance of all filters for the given sensor measurement setting. This is of practical importance in guiding the design and management of sensors to improve target tracking performance. Within the random finite set (RFS) framework, an error bound for joint detection and estimation (JDE) of multiple targets using a single sensor with clutter and missed detection is developed by using multi-Bernoulli or Poisson approximation to multi-target Bayes recursion. Here, JDE refers to jointly estimating the number and states of targets from a sequence of sensor measurements. In order to obtain the results of this paper, all detectors and estimators are restricted to maximum a posteriori (MAP) detectors and unbiased estimators, and the second-order optimal sub-pattern assignment (OSPA) distance is used to measure the error metric between the true and estimated state sets. The simulation results show that clutter density and detection probability have significant impact on the error bound, and the effectiveness of the proposed bound is verified by indicating the performance limitations of the single-sensor probability hypothesis density (PHD) and cardinalized PHD (CPHD) filters for various clutter densities and detection probabilities. PMID:26828499

  14. Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

    PubMed Central

    Hauptmann, Giselbert; Söll, Iris; Krautz, Robert; Theopold, Ulrich

    2016-01-01

    To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos. PMID:26862978

  15. TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models.

    PubMed

    Yao, Zhi-Jiang; Dong, Jie; Che, Yu-Jing; Zhu, Min-Feng; Wen, Ming; Wang, Ning-Ning; Wang, Shan; Lu, Ai-Ping; Cao, Dong-Sheng

    2016-05-01

    Drug-target interactions (DTIs) are central to current drug discovery processes and public health fields. Analyzing the DTI profiling of the drugs helps to infer drug indications, adverse drug reactions, drug-drug interactions, and drug mode of actions. Therefore, it is of high importance to reliably and fast predict DTI profiling of the drugs on a genome-scale level. Here, we develop the TargetNet server, which can make real-time DTI predictions based only on molecular structures, following the spirit of multi-target SAR methodology. Naïve Bayes models together with various molecular fingerprints were employed to construct prediction models. Ensemble learning from these fingerprints was also provided to improve the prediction ability. When the user submits a molecule, the server will predict the activity of the user's molecule across 623 human proteins by the established high quality SAR model, thus generating a DTI profiling that can be used as a feature vector of chemicals for wide applications. The 623 SAR models related to 623 human proteins were strictly evaluated and validated by several model validation strategies, resulting in the AUC scores of 75-100 %. We applied the generated DTI profiling to successfully predict potential targets, toxicity classification, drug-drug interactions, and drug mode of action, which sufficiently demonstrated the wide application value of the potential DTI profiling. The TargetNet webserver is designed based on the Django framework in Python, and is freely accessible at http://targetnet.scbdd.com . PMID:27167132

  16. Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

    NASA Astrophysics Data System (ADS)

    Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

    2012-03-01

    MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve

  17. Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat.

    PubMed

    Carrieri, Claudia; Cimatti, Laura; Biagioli, Marta; Beugnet, Anne; Zucchelli, Silvia; Fedele, Stefania; Pesce, Elisa; Ferrer, Isidre; Collavin, Licio; Santoro, Claudio; Forrest, Alistair R R; Carninci, Piero; Biffo, Stefano; Stupka, Elia; Gustincich, Stefano

    2012-11-15

    Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

  18. COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures.

    PubMed

    Hawkes, Emily J; Hennelly, Scott P; Novikova, Irina V; Irwin, Judith A; Dean, Caroline; Sanbonmatsu, Karissa Y

    2016-09-20

    There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription. PMID:27653675

  19. Does Active Learning through an Antisense Jigsaw Make Sense?

    NASA Astrophysics Data System (ADS)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

    Three journal articles on nucleic acid antisense modification strategies were assigned to 12 students as part of an active learning "jigsaw" exercise for a graduate-level chemistry course on nucleic acids. Each student was required to read one of the three articles. This assignment was preceded by an hour-long lecture on the basic concepts in antisense antigene technology. On the day of the jigsaw, the students with the same article (three groups of four students) discussed their article briefly, and then formed four new groups where no one had read the same article. Each student spent about five minutes teaching his or her article to the other group members, using specific questions provided to guide the discussion. This exercise laid the foundation for bringing the discussion to the entire class, where most of the students actively participated. To test the students' comprehension of the reading materials, a problem set was designed that required not only an understanding of the three articles, but also application of the concepts learned. The effectiveness of this active learning strategy and its applicability to other topics are discussed in this article.

  20. MYCNOS functions as an antisense RNA regulating MYCN

    PubMed Central

    Vadie, Nadia; Saayman, Sheena; Lenox, Alexandra; Ackley, Amanda; Clemson, Mathew; Burdach, Jon; Hart, Jonathan; Vogt, Peter K; Morris, Kevin V

    2015-01-01

    Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci. PMID:26156430

  1. Antisense Reduction of Tau in Adult Mice Protects against Seizures

    PubMed Central

    DeVos, Sarah L.; Goncharoff, Dustin K.; Chen, Guo; Kebodeaux, Carey S.; Yamada, Kaoru; Stewart, Floy R.; Schuler, Dorothy R.; Maloney, Susan E.; Wozniak, David F.; Rigo, Frank; Bennett, C. Frank; Cirrito, John R.; Holtzman, David M.

    2013-01-01

    Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS—brain and spinal cord tissue, interstitial fluid, and CSF—while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability. PMID:23904623

  2. Photoregulating RNA digestion using azobenzene linked dumbbell antisense oligodeoxynucleotides.

    PubMed

    Wu, Li; He, Yujian; Tang, Xinjing

    2015-06-17

    Introduction of 4,4'-bis(hydroxymethyl)-azobenzene (azo) to dumbbell hairpin oligonucleotides at the loop position was able to reversibly control the stability of the whole hairpin structure via UV or visible light irradiation. Here, we designed and synthesized a series of azobenzene linked dumbbell antisense oligodeoxynucleotides (asODNs) containing two terminal hairpins that are composed of an asODN and a short inhibitory sense strand. Thermal melting studies of these azobenzene linked dumbbell asODNs indicated that efficient trans to cis photoisomerization of azobenzene moieties induced large difference in thermal stability (ΔTm = 12.1-21.3 °C). In addition, photomodulation of their RNA binding abilities and RNA digestion by RNase H was investigated. The trans-azobenzene linked asODNs with the optimized base pairs between asODN strands and inhibitory sense strands could only bind few percentage of the target RNA, while it was able to recover their binding to the target RNA and degrade it by RNase H after light irradiation. Upon optimization, it is promising to use these azobenzene linked asODNs for reversible spatial and temporal regulation of antisense activities based on both steric binding and RNA digestion by RNase H.

  3. Artificial neural network prediction of antisense oligodeoxynucleotide activity.

    PubMed

    Giddings, Michael C; Shah, Atul A; Freier, Sue; Atkins, John F; Gesteland, Raymond F; Matveeva, Olga V

    2002-10-01

    An mRNA transcript contains many potential antisense oligodeoxynucleotide target sites. Identification of the most efficacious targets remains an important and challenging problem. Building on separate work that revealed a strong correlation between the inclusion of short sequence motifs and the activity level of an oligo, we have developed a predictive artificial neural network system for mapping tetranucleotide motif content to antisense oligo activity. Trained for high-specificity prediction, the system has been cross-validated against a database of 348 oligos from the literature and a larger proprietary database of 908 oligos. In cross- validation tests the system identified effective oligos (i.e. oligos capable of reducing target mRNA expression to <25% that of the control) with 53% accuracy, in contrast to the <10% success rates commonly reported for trial-and-error oligo selection, suggesting a possible 5-fold reduction in the in vivo screening required to find an active oligo. We have implemented a web interface to a trained neural network. Given an RNA transcript as input, the system identifies the most likely oligo targets and provides estimates of the probabilities that oligos targeted against these sites will be effective. PMID:12364609

  4. Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice

    PubMed Central

    Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Nakatani, Moeka; Wada, Shunsuke; Yasuhara, Hidenori; Narukawa, Keisuke; Sasaki, Kiyomi; Shibata, Masa-Aki; Torigoe, Hidetaka; Yamaoka, Tetsuji; Imanishi, Takeshi; Obika, Satoshi

    2012-01-01

    Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia. PMID:23344002

  5. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  6. Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

    PubMed Central

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

    2013-01-01

    The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  7. Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

    PubMed

    Yu, Xing Xian; Watts, Lynnetta M; Manchem, Vara Prasad; Chakravarty, Kaushik; Monia, Brett P; McCaleb, Michael L; Bhanot, Sanjay

    2013-01-01

    Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

  8. rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus

    PubMed Central

    Shpiz, Sergey; Kwon, Dmitry; Rozovsky, Yakov; Kalmykova, Alla

    2009-01-01

    Telomeres in Drosophila are maintained by the specialized telomeric retrotransposons HeT-A, TART and TAHRE. Sense transcripts of telomeric retroelements were shown to be the targets of a specialized RNA-interference mechanism, a repeat-associated short interfering (rasi)RNA-mediated system. Antisense rasiRNAs play a key role in this mechanism, highlighting the importance of antisense expression in retrotransposon silencing. Previously, bidirectional transcription was reported for the telomeric element TART. Here, we show that HeT-A is also bidirectionally transcribed, and HeT-A antisense transcription in ovaries is regulated by a promoter localized within its 3′ untranslated region. A remarkable feature of noncoding HeT-A antisense transcripts is the presence of multiple introns. We demonstrate that sense and antisense HeT-A-specific rasiRNAs are present in the same tissue, indicating that transcripts of both directions may be considered as natural targets of the rasiRNA pathway. We found that the expression of antisense transcripts of telomeric elements is regulated by the RNA silencing machinery, suggesting rasiRNA-mediated interplay between sense and antisense transcripts in the cell. Finally, this regulation occurs in the nucleus since disruption of the rasiRNA pathway leads to an accumulation of TART and HeT-A transcripts in germ cell nuclei. PMID:19036789

  9. Functional analysis of polyphenol oxidases by antisense/sense technology.

    PubMed

    Thipyapong, Piyada; Stout, Michael J; Attajarusit, Jutharat

    2007-01-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting

  10. A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

    PubMed

    Nishizawa-Yokoi, Ayako; Nonaka, Satoko; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

    2015-09-01

    Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species. PMID:26143254

  11. Antisense oligonucleotide for tissue factor inhibits hepatic ischemic reperfusion injury.

    PubMed

    Nakamura, Kenji; Kadotani, Yayoi; Ushigome, Hidetaka; Akioka, Kiyokazu; Okamoto, Masahiko; Ohmori, Yoshihiro; Yaoi, Takeshi; Fushiki, Shinji; Yoshimura, Rikio; Yoshimura, Norio

    2002-09-27

    Tissue factor (TF) is an initiation factor for blood coagulation and its expression is induced on endothelial cells during inflammatory or immune responses. We designed an antisense oligodeoxynucleotide (AS-1/TF) for rat TF and studied its effect on hepatic ischemic reperfusion injury. AS-1/TF was delivered intravenously to Lewis rats. After 10 h, hepatic artery and portal vein were partially clamped. Livers were reperfused after 180 min and harvested. TF expression was studied using immunohistochemical staining. One of 10 rats survived in a 5-day survival rate and TF was strongly stained on endothelial cells in non-treatment group. However, by treatment with AS-1/TF, six of seven survived and TF staining was significantly reduced. Furthermore, we observed that fluorescein-labeled AS-1/TF was absorbed into endothelial cells. These results suggest that AS-1/TF can strongly suppress the expression of TF and thereby inhibit ischemic reperfusion injury to the rat liver. PMID:12270110

  12. Quantitative Microinjection of Morpholino Antisense Oligonucleotides into Mouse Oocytes to Examine Gene Function in Meiosis-I.

    PubMed

    Nakagawa, Shoma; FitzHarris, Greg

    2016-01-01

    Specific protein depletion is a powerful approach for assessing individual gene function in cellular processes, and has been extensively employed in recent years in mammalian oocyte meiosis-I. Conditional knockout mice and RNA interference (RNAi) methods such as siRNA or dsRNA microinjection are among several approaches to have been applied in this system over the past decade. RNAi by microinjection of Morpholino antisense Oligonucleotides (MO), in particular, has proven highly popular and tractable in many studies, since MOs have high specificity of interaction, low cell toxicity, and are more stable than other microinjected RNAi molecules. Here, we describe a method of MO microinjection into the mouse germinal vesicle-stage (GV) oocyte followed by a simple immunofluorescence approach for examination of gene function in meiosis-I. PMID:27557584

  13. Mutually exclusive sense–antisense transcription at FLC facilitates environmentally induced gene repression

    PubMed Central

    Rosa, Stefanie; Duncan, Susan; Dean, Caroline

    2016-01-01

    Antisense transcription through genic regions is pervasive in most genomes; however, its functional significance is still unclear. We are studying the role of antisense transcripts (COOLAIR) in the cold-induced, epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC), a regulator of the transition to reproduction. Here we use single-molecule RNA FISH to address the mechanistic relationship of FLC and COOLAIR transcription at the cellular level. We demonstrate that while sense and antisense transcripts can co-occur in the same cell they are mutually exclusive at individual loci. Cold strongly upregulates COOLAIR transcription in an increased number of cells and through the mutually exclusive relationship facilitates shutdown of sense FLC transcription in cis. COOLAIR transcripts form dense clouds at each locus, acting to influence FLC transcription through changed H3K36me3 dynamics. These results may have general implications for other loci showing both sense and antisense transcription. PMID:27713408

  14. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides

    PubMed Central

    Lennox, Kim A.; Behlke, Mark A.

    2016-01-01

    Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. PMID:26578588

  15. Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

    PubMed

    Moschos, Sterghios A; Frick, Manfred; Taylor, Bruce; Turnpenny, Paul; Graves, Helen; Spink, Karen G; Brady, Kevin; Lamb, David; Collins, David; Rockel, Thomas D; Weber, Markus; Lazari, Ovadia; Perez-Tosar, Luis; Fancy, Sally A; Lapthorn, Chris; Green, Martin X; Evans, Steve; Selby, Matthew; Jones, Gareth; Jones, Lyn; Kearney, Sarah; Mechiche, Houria; Gikunju, Diana; Subramanian, Romesh; Uhlmann, Eugen; Jurk, Marion; Vollmer, Jörg; Ciaramella, Giuseppe; Yeadon, Michael

    2011-12-01

    Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

  16. Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides.

    PubMed

    Lennox, Kim A; Behlke, Mark A

    2016-01-29

    Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs (TUG1, CasC7 and HOTAIR) were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. PMID:26578588

  17. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  18. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    PubMed

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  19. Natural Antisense Transcripts and Long Non-Coding RNA in Neurospora crassa

    PubMed Central

    Arthanari, Yamini; Heintzen, Christian; Griffiths-Jones, Sam; Crosthwaite, Susan K.

    2014-01-01

    The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs. PMID:24621812

  20. Advanced In vivo Use of CRISPR/Cas9 and Anti-sense DNA Inhibition for Gene Manipulation in the Brain

    PubMed Central

    Walters, Brandon J.; Azam, Amber B.; Gillon, Colleen J.; Josselyn, Sheena A.; Zovkic, Iva B.

    2016-01-01

    Gene editing tools are essential for uncovering how genes mediate normal brain–behavior relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene editing technology now allows neuroscientists unprecedented access to edit the genome efficiently. Although many important tools have been developed, here we focus on approaches that allow for rapid gene editing in the adult nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques. CRISPR/Cas9 is a flexible gene editing tool, allowing the genome to be manipulated in diverse ways. For instance, CRISPR/Cas9 has been successfully used to knockout genes, knock-in mutations, overexpress or inhibit gene activity, and provide scaffolding for recruiting specific epigenetic regulators to individual genes and gene regions. Moreover, the CRISPR/Cas9 system may be modified to target multiple genes at one time, affording simultaneous inhibition and overexpression of distinct genetic targets. Although many of the more advanced applications of CRISPR/Cas9 have not been applied to the nervous system, the toolbox is widely accessible, such that it is poised to help advance neuroscience. Anti-sense nucleotide-based technologies can be used to rapidly knockdown genes in the brain. The main advantage of anti-sense based tools is their simplicity, allowing for rapid gene delivery with minimal technical expertise. Here, we describe the main applications and functions of each of these systems with an emphasis on their many potential applications in neuroscience laboratories. PMID:26793235

  1. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    PubMed

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  2. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

    PubMed

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-01-01

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species. PMID:27646849

  3. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells

    PubMed Central

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-01-01

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5′ UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3′ end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species. PMID:27646849

  4. Autoregulatory loop of Msx1 expression involving its antisense transcripts.

    PubMed

    Petit, Stéphane; Meary, Fleur; Pibouin, Laurence; Jeanny, Jean-Claude; Fernandes, Isabelle; Poliard, Anne; Hotton, Dominique; Berdal, Ariane; Babajko, Sylvie

    2009-08-01

    The Msx1 homeogene plays an important role in epithelial-mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis.

  5. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides

    PubMed Central

    Wang, Shiyu; Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2016-01-01

    Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release. PMID:27378781

  6. Biodistribution of antisense nanoparticles in mammary carcinoma rat model.

    PubMed

    Elazar, Victoria; Adwan, Hassan; Rohekar, Keren; Zepp, Michael; Lifshitz-Shovali, Rinat; Berger, Martin R; Golomb, Gershon

    2010-08-01

    Efficient and specific delivery of antisenses (ASs) and protection of the sequences from degradation are critical factors for effective therapy. Sustained release nanoparticles (NP) offer increased resistance to nuclease degradation, increased amounts of AS uptake, and the possibility of control in dosing and sustained duration of AS administration. The biodegradable and biocompatible poly(D,L-lactic-co-glycolic acid) copolymer (PLGA) was utilized to encapsulate AS directed against osteopontin (OPN), which is a promising therapeutic target in mammary carcinoma. Whole body biodistribution of OPN AS NP was evaluated in comparison to naked AS, in intact and mammary carcinoma metastasis model bearing rats. Naked and NP encapsulated AS exhibited different biodistribution profiles. AS NP, in contrast to naked AS, tended to accumulate mostly in the spleen, liver, and at the tumor inoculation site. Drug levels in intact organs were negligible. The elimination of naked AS was faster, due to rapid degradation of the unprotected sequence. It is concluded that AS NP protect the AS from degradation, provide efficient AS delivery to the tumor tissue, and minimize AS accumulation in intact organs due to the AS sustained release profile as well as the favorable NP physicochemical properties.

  7. Antisense properties of duplex- and triplex-forming PNAs.

    PubMed Central

    Knudsen, H; Nielsen, P E

    1996-01-01

    The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA. PMID:8602363

  8. Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

    PubMed Central

    Lenartowicz, Elzbieta; Nogales, Aitor; Kierzek, Elzbieta; Kierzek, Ryszard; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches. PMID:27463680

  9. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    PubMed

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  10. Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli.

    PubMed

    Thomason, Maureen K; Bischler, Thorsten; Eisenbart, Sara K; Förstner, Konrad U; Zhang, Aixia; Herbig, Alexander; Nieselt, Kay; Sharma, Cynthia M; Storz, Gisela

    2015-01-01

    While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.

  11. Sequence motifs associated with hepatotoxicity of locked nucleic acid—modified antisense oligonucleotides

    PubMed Central

    Burdick, Andrew D.; Sciabola, Simone; Mantena, Srinivasa R.; Hollingshead, Brett D.; Stanton, Robert; Warneke, James A.; Zeng, Ming; Martsen, Elena; Medvedev, Alexander; Makarov, Sergei S.; Reed, Lori A.; Davis, John W.; Whiteley, Laurence O.

    2014-01-01

    Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing. PMID:24550163

  12. Intravesical Liposome and Antisense Treatment for Detrusor Overactivity and Interstitial Cystitis/Painful Bladder Syndrome

    PubMed Central

    Kashyap, Mahendra P.; Kawamorita, Naoki; Yoshizawa, Tsuyoshi; Chancellor, Michael

    2014-01-01

    Purpose. The following review focuses on the recent advancements in intravesical drug delivery, which brings added benefit to the therapy of detrusor overactivity and interstitial cystitis/painful bladder syndrome (IC/PBS). Results. Intravesical route is a preferred route of administration for restricting the action of extremely potent drugs like DMSO for patients of interstitial cystitis/painful bladder syndrome (IC/PBS) and botulinum toxin for detrusor overactivity. Patients who are either refractory to oral treatment or need to mitigate the adverse effects encountered with conventional routes of administration also chose this route. Its usefulness in some cases can be limited by vehicle (carrier) toxicity or short duration of action. Efforts have been underway to overcome these limitations by developing liposome platform for intravesical delivery of biotechnological products including antisense oligonucleotides. Conclusions. Adoption of forward-thinking approaches can achieve advancements in drug delivery systems targeted to future improvement in pharmacotherapy of bladder diseases. Latest developments in the field of nanotechnology can bring this mode of therapy from second line of treatment for refractory cases to the forefront of disease management. PMID:24527221

  13. Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica

    PubMed Central

    Morf, Laura; Pearson, Richard J.; Wang, Angelia S.; Singh, Upinder

    2013-01-01

    RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects. PMID:23935116

  14. Gene expression analysis by a competitive and differential PCR with antisense competitors.

    PubMed

    de Kant, E; Rochlitz, C F; Herrmann, R

    1994-11-01

    We report a sensitive method for the reproducible and accurate measurement of gene expression from small samples of RNA. This method is based on a combination of two PCR techniques: First, an endogenous reporter gene and the gene of interest are simultaneously amplified in one tube after random-primed reverse transcription (RT) of RNA (differential RT-PCR). Second, exogenous homologous fragments of both genes with artificially introduced mutations are added and coamplified in the same reaction (competitive PCR). The first-strand cDNA, and the mutated antisense homologues of the reporter as well as the target gene compete for their respective primers and are therefore amplified with equal efficiencies. After PCR, restriction enzyme digestion allows visualization of the quantitative differences between the four resulting reaction products. The ratios of products that competed during PCR provide the quantitative information. The initial amount of a specific cDNA can be calculated from any competitor/cDNA ratio of reliably measurable PCR product amounts. Extensive competitor titration to experimentally approach the equilibrium is therefore unnecessary. The differential counterpart of competitive and differential RT-PCR (CD-RT-PCR) allows expression of the levels in reference to a reporter gene. MDR1 expression was determined in tumor cells by CD-RT-PCR.

  15. PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

    NASA Astrophysics Data System (ADS)

    Azeke, John Imuetinyan-Jesu, Jr.

    , while both cytokines are over-expressed within the first day following injury, CTGF protein levels could not be correlated with observed adhesion development. In addition, we synthesized linear triblock copolymers of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA), two of the most widely studied biodegradable polymers in use today. Bulk gels and microparticles of the copolymers were then evaluated for gelling behavior, temperature stability, and drug loading and release kinetics in order assess their suitability as potential carriers of antisense therapeutics. A novel approach to affecting the antisense oligonucleotide release kinetics by varying the relative concentrations of co-encapsulated cationic lipid transfection agents was also presented.

  16. Intracerebroventricular Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuates Salt Appetite in the Rat.

    PubMed

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

    The anterior ventral third ventricle (AV3V) region of the brain contains high concentrations of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) that are important in the maintenance of body fluid and electrolyte balance as well as other physiological processes. Daily intracerebroventricular pulse injections of MR antisense oligonucleotides significantly suppressed deoxycorticosterone acetate (DOCA) induced salt appetite in a dose-related manner. Similar administration of GR antisense or scrambled/sense oligonucleotide into the third ventricle failed to inhibit salt appetite. Salt appetite aroused after adrenalectomy was not suppressed by MR antisense oligonucleotide treatments but was suppressed by an antisense oligonucleotide directed against the angiotensin II AT1 receptor subtype. Receptor binding analysis demonstrated that MR and GR oligonucleotide treatments each reduced their respective receptor subtypes. Finally, although GR antisense oligonucleotide treatment was ineffective in suppressing DOCA-induced salt appetite, this treatment did increase stress induced corticosterone release as well as delayed the recovery of corticosterone to basal levels after stress. PMID:9787254

  17. Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

    PubMed Central

    Krieg, A M; Tonkinson, J; Matson, S; Zhao, Q; Saxon, M; Zhang, L M; Bhanja, U; Yakubov, L; Stein, C A

    1993-01-01

    Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides. Images PMID:8430072

  18. Prediction of Multi-Target Networks of Neuroprotective Compounds with Entropy Indices and Synthesis, Assay, and Theoretical Study of New Asymmetric 1,2-Rasagiline Carbamates

    PubMed Central

    Romero Durán, Francisco J.; Alonso, Nerea; Caamaño, Olga; García-Mera, Xerardo; Yañez, Matilde; Prado-Prado, Francisco J.; González-Díaz, Humberto

    2014-01-01

    In a multi-target complex network, the links (Lij) represent the interactions between the drug (di) and the target (tj), characterized by different experimental measures (Ki, Km, IC50, etc.) obtained in pharmacological assays under diverse boundary conditions (cj). In this work, we handle Shannon entropy measures for developing a model encompassing a multi-target network of neuroprotective/neurotoxic compounds reported in the CHEMBL database. The model predicts correctly >8300 experimental outcomes with Accuracy, Specificity, and Sensitivity above 80%–90% on training and external validation series. Indeed, the model can calculate different outcomes for >30 experimental measures in >400 different experimental protocolsin relation with >150 molecular and cellular targets on 11 different organisms (including human). Hereafter, we reported by the first time the synthesis, characterization, and experimental assays of a new series of chiral 1,2-rasagiline carbamate derivatives not reported in previous works. The experimental tests included: (1) assay in absence of neurotoxic agents; (2) in the presence of glutamate; and (3) in the presence of H2O2. Lastly, we used the new Assessing Links with Moving Averages (ALMA)-entropy model to predict possible outcomes for the new compounds in a high number of pharmacological tests not carried out experimentally. PMID:25255029

  19. Searching for Multi-Targeting Neurotherapeutics against Alzheimer's: Discovery of Potent AChE-MAO B Inhibitors through the Decoration of the 2H-Chromen-2-one Structural Motif.

    PubMed

    Pisani, Leonardo; Farina, Roberta; Soto-Otero, Ramon; Denora, Nunzio; Mangiatordi, Giuseppe Felice; Nicolotti, Orazio; Mendez-Alvarez, Estefania; Altomare, Cosimo Damiano; Catto, Marco; Carotti, Angelo

    2016-03-17

    The need for developing real disease-modifying drugs against neurodegenerative syndromes, particularly Alzheimer's disease (AD), shifted research towards reliable drug discovery strategies to unveil clinical candidates with higher therapeutic efficacy than single-targeting drugs. By following the multi-target approach, we designed and synthesized a novel class of dual acetylcholinesterase (AChE)-monoamine oxidase B (MAO-B) inhibitors through the decoration of the 2H-chromen-2-one skeleton. Compounds bearing a propargylamine moiety at position 3 displayed the highest in vitro inhibitory activities against MAO-B. Within this series, derivative 3h emerged as the most interesting hit compound, being a moderate AChE inhibitor (IC50 = 8.99 µM) and a potent and selective MAO-B inhibitor (IC50 = 2.8 nM). Preliminary studies in human neuroblastoma SH-SY5Y cell lines demonstrated its low cytotoxicity and disclosed a promising neuroprotective effect at low doses (0.1 µM) under oxidative stress conditions promoted by two mitochondrial toxins (oligomycin-A and rotenone). In a Madin-Darby canine kidney (MDCK)II-MDR1 cell-based transport study, Compound 3h was able to permeate the BBB-mimicking monolayer and did not result in a glycoprotein-p (P-gp) substrate, showing an efflux ratio = 0.96, close to that of diazepam.

  20. Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

    PubMed Central

    2012-01-01

    Background Cis-natural antisense transcripts (cis-NATs) are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs), which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq) to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa). Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set), 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions Our study profiles an

  1. Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA.

    PubMed

    Bendahmane, M; Gronenborn, B

    1997-01-01

    One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny. PMID:9037152

  2. Antisense overlapping open reading frames in genes from bacteria to humans.

    PubMed Central

    Merino, E; Balbás, P; Puente, J L; Bolívar, F

    1994-01-01

    Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far. PMID:8208617

  3. A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

    PubMed

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  4. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast

    PubMed Central

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y.; Brem, Rachel B.

    2016-01-01

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1. Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1. Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them. PMID:27190003

  5. Use of an Antisense RNA Strategy To Investigate the Functional Significance of Mn-Catalase in the Extreme Thermophile Thermus thermophilus

    PubMed Central

    Moreno, Renata; Hidalgo, Aurelio; Cava, Felipe; Fernández-Lafuente, Roberto; Guisán, José Manuel; Berenguer, José

    2004-01-01

    The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a “bicistronic” transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target. PMID:15516595

  6. Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-beta1 inhibits fibrogenic activities of hepatic stellate cells.

    PubMed

    Arias, Monica; Lahme, Birgit; Van de Leur, Eddy; Gressner, Axel M; Weiskirchen, Ralf

    2002-06-01

    Liver fibrosis occurs as a consequence of the transdifferentiationof hepatic stellate cells into myofibroblasts and is associated with an increased expression and activation of transforming growth factor (TGF)-beta1. This pluripotent, profibrogenic cytokine stimulates matrix synthesis and decreases matrix degradation, resulting in fibrosis. Thus, blockade of synthesis or sequestering of mature TGF-beta1 is a primary target for the development of antifibrotic approaches. The purpose of this study was to investigate whether the administration of adenoviruses constitutively expressing an antisense mRNA complementary to the 3' coding sequence of TGF-beta1 is able to suppress the synthesis of TGF-beta1 in culture-activated hepatic stellate cells. We demonstrate that the adenoviral vehicle directs high-level expression of the transgene and proved that the transduced antisense is biologically active by immunoprecipitation, Western blot, quantitative TGF-beta1 ELISA, and cell proliferation assays. Additionally, the biological function of the transgene was confirmed by analysis of differential activity of TGF-beta1-responsive genes using cell ELISA, Northern blotting, and by microarray technology, respectively. Furthermore, we examined the effects of that transgene on the expression of TGF-beta2, TGF-beta3, collagen type alpha1(I), latent transforming growth factor binding protein 1, types I and II TGF-beta receptors, and alpha-smooth muscle actin. Our results indicate that the administration of antisense mRNA offers a feasible approach to block autocrine TGF-beta1 signaling in hepatic stellate cells and may be useful and applicable in future to the treatment of fibrosis in chronic liver diseases.

  7. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

    PubMed Central

    Lagier-Tourenne, Clotilde; Baughn, Michael; Rigo, Frank; Sun, Shuying; Liu, Patrick; Li, Hai-Ri; Jiang, Jie; Watt, Andrew T.; Chun, Seung; Katz, Melanie; Qiu, Jinsong; Sun, Ying; Ling, Shuo-Chien; Zhu, Qiang; Polymenidou, Magdalini; Drenner, Kevin; Artates, Jonathan W.; McAlonis-Downes, Melissa; Markmiller, Sebastian; Hutt, Kasey R.; Pizzo, Donald P.; Cady, Janet; Harms, Matthew B.; Baloh, Robert H.; Vandenberg, Scott R.; Yeo, Gene W.; Fu, Xiang-Dong; Bennett, C. Frank; Cleveland, Don W.; Ravits, John

    2013-01-01

    Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions. PMID:24170860

  8. Delivery of antisense oligonucleotide to the cornea by iontophoresis.

    PubMed

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F

    2003-04-01

    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  9. The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.

    PubMed

    Edwards, Mary E; Choo, Tze-Siang; Dickson, Cathryn A; Scott, Catherine; Gidley, Michael J; Reid, J S Grant

    2004-03-01

    Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

  10. Synthesis of Thiazolo[5,4-f]quinazolin-9(8H)-ones as Multi-Target Directed Ligands of Ser/Thr Kinases.

    PubMed

    Hédou, Damien; Godeau, Julien; Loaëc, Nadège; Meijer, Laurent; Fruit, Corinne; Besson, Thierry

    2016-01-01

    A library of thirty novel thiazolo[5,4-f]quinazolin-9(8H)-one derivatives belonging to four series designated as 12, 13, 14 and 15 was efficiently prepared, helped by microwave-assisted technology when required. The efficient multistep synthesis of methyl 6-amino-2-cyano- benzo[d]thiazole-7-carboxylate (1) has been reinvestigated and performed on a multigram scale. The inhibitory potency of the final products against five kinases involved in Alzheimer's disease was evaluated. This study demonstrates that some molecules of the 12 and 13 series described in this paper are particularly promising for the development of new multi-target inhibitors of kinases. PMID:27144552

  11. Multi-targeting Peptide-Functionalized Nanoparticles Recognized Vasculogenic Mimicry, Tumor Neovasculature, and Glioma Cells for Enhanced Anti-glioma Therapy.

    PubMed

    Feng, Xingye; Yao, Jianhui; Gao, Xiaoling; Jing, Yixian; Kang, Ting; Jiang, Di; Jiang, Tianze; Feng, Jingxian; Zhu, Qianqian; Jiang, Xinguo; Chen, Jun

    2015-12-23

    Chemotherapy failure of glioma, the most aggressive and devastating cancer, might be ascribed to the physiologic barriers of the tumor mainly including heterogeneous tumor perfusion and vascular permeability, which result in a limited penetration of chemotherapeutics. Besides, the vasculogenic mimicry (VM) channels, which are highly resistant to anti-angiogenic therapy and serve as a complement of angiogenesis, were abound in glioma and always associated with tumor recurrence. In order to enhance the therapy effect of anti-glioma, we developed a PEG-PLA-based nanodrug delivery system (nanoparticles, NP) in this study and modified its surface with CK peptide, which was composed of a human sonic hedgehog (SHH) targeting peptide (CVNHPAFAC) and a KDR targeting peptide (K237) through a GYG linker, for facilitating efficient VM channels, tumor neovasculature, and glioma cells multi-targeting delivery of paclitaxel. In vitro cellular assay showed that CK-NP-PTX not only exhibited the strongest antiproliferation effect on U87MG cells and HUVEC cells but also resulted in the most efficient destruction of VM channels when compared with CVNHPAFAC-NP, K237-NP, and the unmodified ones. Besides, CK-NP accumulated more selectively at the glioma site as demonstrated by in vivo and ex vivo imaging. As expected, the glioma-bearing mice treated with CK-NP-PTX achieved the longest median survival time compared to those treated with CVNHPAFAC-NP-PTX and K237-NP-PTX. These findings indicated that the multi-targeting therapy mediated by CK peptide might provide a promising way for glioblastoma therapy.

  12. Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

    PubMed

    Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

    2010-01-01

    Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

  13. Impact of DNA gyrase inhibition by antisense ribozymes on rec A in E. coli.

    PubMed

    Shilpakala, Sainath Rao; Raghunathan, Malathi

    2009-09-01

    The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluoroquinolones.

  14. Nanoparticle-Delivered Antisense MicroRNA-21 Enhances the Effects of Temozolomide on Glioblastoma Cells.

    PubMed

    Ananta, Jeyarama S; Paulmurugan, Ramasamy; Massoud, Tarik F

    2015-12-01

    Glioblastoma (GBM) generally exhibits high IC50 values for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21) is an oncomiR overexpressed in GBM, thus controlling important aspects of glioma biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 to glioblastoma cells might beneficially knock down endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating antisense miR-21 were effective in intracellular delivery and sustained silencing (p < 0.01) of miR-21 function in U87 MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly reduced the number of viable cells (p < 0.001), and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ treatment in U87 MG cells. There was overexpression of the miR-21 target genes PTEN (by 67%) and caspase-3 (by 15%) upon cotreatment. This promising PLGA nanoparticle-based platform for antisense miR-21 delivery to GBM is an effective cotherapeutic strategy in cell culture, warranting the need for further studies prior to future clinical translation. PMID:26559642

  15. An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

    PubMed Central

    Subudhi, Amit Kumar; Boopathi, P.A.; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B.; Mugasimangalam, Raja C.; Sirohi, Paramendra; Kochar, Sanjay K.; Kochar, Dhanpat K.; Das, Ashis

    2014-01-01

    Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014). PMID:26484136

  16. Potent Antibacterial Antisense Peptide–Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    PubMed Central

    Ghosal, Anubrata

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce antisense peptide–peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide–PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)4-Ahx-βala or the H-(R-Ahx)6-βala peptide exhibited complete growth inhibition of P. aeruginosa strains PA01, PA14, and LESB58 at 1–2 μM concentrations without any indication of bacterial membrane disruption (even at 20 μM), and resulted in specific reduction of the targeted mRNA levels. One of the four compounds showed clear bactericidal activity while the other significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections. PMID:23030590

  17. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

    PubMed

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-03-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

  18. Drug evaluation: ISIS-301012, an antisense oligonucleotide for the treatment of hypercholesterolemia.

    PubMed

    Burnett, John R

    2006-10-01

    ISIS-301012 is an antisense oligonucleotide inhibitor of apolipoprotein B-100, which is being developed by Isis Pharmaceuticals Inc for the potential treatment of hypercholesterolemia. A subcutaneous injectable formulation is currently undergoing phase 11 clinical trials, while phase I trials are underway with an oral formulation of the drug.

  19. Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription.

    PubMed

    Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J; van Hooff, Sander; van Leenen, Dik; Dargemont, Catherine; Géli, Vincent; Holstege, Frank C P

    2012-09-01

    Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by

  20. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  1. Interaction of α-melanocortin and its pentapeptide antisense LVKAT: effects on hepatoprotection in male CBA mice.

    PubMed

    Houra, Karlo; Turčić, Petra; Gabričević, Mario; Weitner, Tin; Konjevoda, Paško; Stambuk, Nikola

    2011-01-01

    The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of continuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3'→5' direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo. PMID:21873934

  2. Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

    PubMed

    Shen, Z J; Kim, S K; Kwon, O S; Lee, Y S; Moon, B J

    2001-04-01

    To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.

  3. Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon.

    PubMed

    Vijayanathan, Veena; Thomas, Thresia; Sigal, Leonard H; Thomas, T J

    2002-08-01

    A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.

  4. Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma

    PubMed Central

    Andreyev, H; Ross, P; Cunningham, D; Clarke, P

    2001-01-01

    BACKGROUND—Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer.
AIMS—To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction.
METHODS—Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture.
RESULTS—The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively.
CONCLUSIONS—Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA.


Keywords: Ki-ras mutation; antisense treatment; colorectal carcinoma PMID:11156646

  5. Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

    1996-01-01

    By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

  6. Linear decay of retrotransposon antisense bias across genes is contingent upon tissue specificity.

    PubMed

    Linker, Sara; Hedges, Dale

    2013-01-01

    Retrotransposons comprise approximately half of the human genome and contribute to chromatin structure, regulatory motifs, and protein-coding sequences. Since retrotransposon insertions can disrupt functional genetic elements as well as introduce new sequence motifs to a region, they have the potential to affect the function of genes that harbour insertions as well as those nearby. Partly as a result of these effects, the distribution of retrotransposons across the genome is non-uniform and there are observed imbalances in the orientation of insertions with respect to the transcriptional direction of the containing gene. Although some of the factors underlying the observed distributions are understood, much of the variability remains unexplained. Detailed characterization of retrotransposon density in genes could help inform predictions of the functional consequence of de novo as well as polymorphic insertions. In order to characterize the relationship between genes and inserted elements, we have examined the distribution of retrotransposons and their internal motifs within tissue-specific and housekeeping genes. We have identified that the previously established retrotransposon antisense bias decays at a linear rate across genes, resulting in an equal density of sense and antisense retrotransposons near the 3'-UTR. In addition, the decay of antisense bias across genes is less pronounced among tissue-specific genes. Our results provide support for the scenario in which this linear decay in antisense bias is established by natural selection shortly after retrotransposon integration, and that total antisense bias observed is above and beyond any bias introduced by the integration process itself. Finally, we provide an example of a retrotransposon acting as an eQTL on a coincident gene, highlighting one of several possible avenues through which insertions may modulate gene function.

  7. Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

    PubMed Central

    Walton, S Patrick; Stephanopoulos, Gregory N; Yarmush, Martin L; Roth, Charles M

    2002-01-01

    Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed. PMID:11751323

  8. A multi-target approach for pain treatment: dual inhibition of fatty acid amide hydrolase and TRPV1 in a rat model of osteoarthritis.

    PubMed

    Malek, Natalia; Mrugala, Monika; Makuch, Wioletta; Kolosowska, Natalia; Przewlocka, Barbara; Binkowski, Marcin; Czaja, Martyna; Morera, Enrico; Di Marzo, Vincenzo; Starowicz, Katarzyna

    2015-05-01

    The pharmacological inhibition of anandamide (AEA) hydrolysis by fatty acid amide hydrolase (FAAH) attenuates pain in animal models of osteoarthritis (OA) but has failed in clinical trials. This may have occurred because AEA also activates transient receptor potential vanilloid type 1 (TRPV1), which contributes to pain development. Therefore, we investigated the effectiveness of the dual FAAH-TRPV1 blocker OMDM-198 in an MIA-model of osteoarthritic pain. We first investigated the MIA-induced model of OA by (1) characterizing the pain phenotype and degenerative changes within the joint using X-ray microtomography and (2) evaluating nerve injury and inflammation marker (ATF-3 and IL-6) expression in the lumbar dorsal root ganglia of osteoarthritic rats and differences in gene and protein expression of the cannabinoid CB1 receptors FAAH and TRPV1. Furthermore, we compared OMDM-198 with compounds acting exclusively on FAAH or TRPV1. Osteoarthritis was accompanied by the fragmentation of bone microstructure and destroyed cartilage. An increase of the mRNA levels of ATF3 and IL-6 and an upregulation of AEA receptors and FAAH in the dorsal root ganglia were observed. OMDM-198 showed antihyperalgesic effects in the OA model, which were comparable with those of a selective TRPV1 antagonist, SB-366,791, and a selective FAAH inhibitor, URB-597. The effect of OMDM-198 was attenuated by the CB1 receptor antagonist, AM-251, and by the nonpungent TRPV1 agonist, olvanil, suggesting its action as an "indirect" CB1 agonist and TRPV1 antagonist. These results suggest an innovative strategy for the treatment of OA, which may yield more satisfactory results than those obtained so far with selective FAAH inhibitors in human OA.

  9. Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

    PubMed

    Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

    2009-06-01

    Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that

  10. Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

    PubMed Central

    Hanecak, R; Brown-Driver, V; Fox, M C; Azad, R F; Furusako, S; Nozaki, C; Ford, C; Sasmor, H; Anderson, K P

    1996-01-01

    Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and

  11. Suppression of endo B cytokeratin by its antisense RNA inhibits the normal coexpression of endo A cytokeratin.

    PubMed Central

    Trevor, K; Linney, E; Oshima, R G

    1987-01-01

    Antisense endo B cytokeratin RNA encoded by a retrovirus vector was expressed in a derivative of the F9 embryonal carcinoma cell line. Two G418-resistant clones were selected that expressed a colinear transcript containing both neomycin and antisense endo B cytokeratin sequences. Expression of a 5-fold excess of antisense endo B RNA over endogenous, retinoic acid-induced endo B RNA resulted in suppression of endo B cytokeratin protein expression. In addition, the normal induction of endo A protein, the type II cytokeratin that polymerizes with endo B, was suppressed at the RNA and protein levels. Revertant clones, which synthesize little if any neo or antisense endo B RNA, regain the ability to express the affected gene products in response to retinoic acid. These results indicate that the suppression of endo B cytokeratin protein synthesis influences the stable levels of endo A mRNA. Images PMID:2434948

  12. On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23.

    PubMed

    Nicole, L M; Tanguay, R M

    1987-03-01

    The specificity of action of antisense RNA for one of Drosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translation in vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45 degrees C) and low stringency (37 degrees C) conditions. This suggests that the 5' leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

  13. Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes.

    PubMed

    Movassaghian, Sara; Moghimi, Hamid R; Shirazi, Farshad H; Koshkaryev, Alexander; Trivedi, Malav S; Torchilin, Vladimir P

    2013-01-30

    The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques. PMID:23262426

  14. Growth inhibition of DU-145 prostate cancer cells by a Bcl-2 antisense oligonucleotide is enhanced by N-(2-hydroxyphenyl)all-trans retinamide.

    PubMed

    Campbell, M J; Dawson, M; Koeffler, H P

    1998-03-01

    Hormonally insensitive prostate cancer is a relatively slow-growing, but usually fatal, disease with no long-term treatment options. Transformation of normal prostate cells to a malignant phenotype often involves corruption of the apoptotic machineries. Bcl-2 protein is one of the key inhibitors of apoptosis and is often unregulated in advanced prostate cancer. The prostate cancer cell line DU-145 was used as a model of a hormonally insensitive, advanced prostate cancer. Cell growth in liquid culture was significantly inhibited by antisense Bcl-2 oligonucleotides compared with control sense oligonucleotides; inhibition by these oligonucleotides was significantly enhanced on combination with the synthetic retinoid N-(2-hydroxyphenyl)all-trans-retinamide (2-HPR). Interestingly, growth inhibition occurred in the absence of apoptosis as measured using two assay techniques. We hypothesize that in these recalcitrant cells the apoptotic pathway is compromised at several levels, and Bcl-2 may play another role in promoting cell growth. The use of Bcl-2 antisense oligonucleotides plus 2-HPR may provide a novel approach to therapy of hormone-resistant prostate cancer.

  15. Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

    PubMed Central

    Doran, Philip; Wilton, Steve D.; Fletcher, Sue; Ohlendieck, Kay

    2009-01-01

    The disintegration of the dystrophin-glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, β-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+-handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. PMID:19132684

  16. Self-assembled phenylalanine-α,β-dehydrophenylalanine nanotubes for sustained intravitreal delivery of a multi-targeted tyrosine kinase inhibitor.

    PubMed

    Panda, Jiban J; Yandrapu, Sarath; Kadam, Rajendra S; Chauhan, Virander S; Kompella, Uday B

    2013-12-28

    Current standard of care for sustained back of the eye drug delivery is surgical placement or injection of large, slow release implants using a relatively large 22 gauge needle. We designed novel dipeptide (phenylalanine-α,β-dehydrophenylalanine; Phe-∆Phe) based nanotubes with a diameter of ~15-30 nm and a length of ~1500 nm that could be injected with a 33 gauge needle for sustained intravitreal delivery of pazopanib, a multi-targeted tyrosine kinase inhibitor. The drug could be loaded during nanotube assembly or post-loaded after nanotube formation, with the former being more efficient at 25% w/w pazopanib loading and ~55% loading efficiency. Plain and peptide loaded nanotube were non-cytotoxic to retinal pigment epithelial cells even at a concentration of 200 μg/ml. Following intravitreal injection of fluorescently labeled nanotubes using a 33 gauge needle in a rat model, the nanotube persistence and drug delivery were monitored using noninvasive fluorophotometry, electron microscopy and mass spectrometry analysis. Nanotubes persisted in the vitreous humor during the 15 days study and pazopanib levels in the vitreous humor, retina, and choroid-RPE at the end of the study were 4.5, 5, and 2.5-folds higher, respectively, compared to the plain drug. Thus, Phe-∆Phe nanotubes allow intravitreal injections with a small gauge needle and sustain drug delivery.

  17. Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity

    PubMed Central

    Serobyan, Vahan; Xiao, Hua; Namdeo, Suryesh; Rödelsperger, Christian; Sieriebriennikov, Bogdan; Witte, Hanh; Röseler, Waltraud; Sommer, Ralf J.

    2016-01-01

    Phenotypic plasticity has been suggested to act through developmental switches, but little is known about associated molecular mechanisms. In the nematode Pristionchus pacificus, the sulfatase eud-1 was identified as part of a developmental switch controlling mouth-form plasticity governing a predatory versus bacteriovorous mouth-form decision. Here we show that mutations in the conserved histone-acetyltransferase Ppa-lsy-12 and the methyl-binding-protein Ppa-mbd-2 mimic the eud-1 phenotype, resulting in the absence of one mouth-form. Mutations in both genes cause histone modification defects and reduced eud-1 expression. Surprisingly, Ppa-lsy-12 mutants also result in the down-regulation of an antisense-eud-1 RNA. eud-1 and antisense-eud-1 are co-expressed and further experiments suggest that antisense-eud-1 acts through eud-1 itself. Indeed, overexpression of the antisense-eud-1 RNA increases the eud-1-sensitive mouth-form and extends eud-1 expression. In contrast, this effect is absent in eud-1 mutants indicating that antisense-eud-1 positively regulates eud-1. Thus, chromatin remodelling and antisense-mediated up-regulation of eud-1 control feeding plasticity in Pristionchus. PMID:27487725

  18. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    PubMed Central

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  19. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    PubMed

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  20. Alteration of the self-incompatibility phenotype in Brassica by transformation of the antisense SLG gene.

    PubMed

    Shiba, H; Kimura, N; Takayama, S; Hinata, K; Suzuki, A; Isogai, A

    2000-05-01

    Self-incompatible (SI) Brassica rapa (syn. B. campestris) was transformed with an antisense SLG gene by using SLG8 cDNA isolated from the B. campestris S8 homozygote. Two transformed lines were obtained and analyzed. Northern blot and Western blot analyses revealed that endogenous SLG and SRK were greatly reduced of the transcriptional and translational levels in the transformant. Pollination experiments confirmed that their SI phenotype had broken down. In addition, the progeny with the antisense SLG gene, resulting from self- or cross-pollination of the transgenic plant, also showed the self-compatible phenotype. The breakdown of SI in the tranformants was due to the change in property of the stigma and not of the pollen. These results provide strong evidence that SLG and/or SRK is implicated in the pollen-stigma recognition of SI and that they act only as stigmatic factors.

  1. Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B

    PubMed Central

    Crooke, Stanley T; Geary, Richard S

    2013-01-01

    Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50–400 mg week−1, both as a single agent and in the presence of maximal lipid lowering therapy. No drug–drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. PMID:23013161

  2. Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription

    PubMed Central

    Marquardt, Sebastian; Raitskin, Oleg; Wu, Zhe; Liu, Fuquan; Sun, Qianwen; Dean, Caroline

    2014-01-01

    Summary Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms. PMID:24725596

  3. Mechanistic principles of antisense targets for the treatment of spinal muscular atrophy.

    PubMed

    Singh, Natalia N; Lee, Brian M; DiDonato, Christine J; Singh, Ravindra N

    2015-01-01

    Spinal muscular atrophy (SMA) is a major neurodegenerative disorder of children and infants. SMA is primarily caused by low levels of SMN protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of the production of the functional SMN protein due to predominant skipping of exon 7. Several compounds, including antisense oligonucleotides (ASOs) that elevate SMN protein from SMN2 hold the promise for treatment. An ASO-based drug currently under Phase III clinical trial employs intronic splicing silencer N1 (ISS-N1) as its target. Cumulative studies on ISS-N1 reveal a wealth of information with significance to the overall therapeutic development for SMA. Here, the authors summarize the mechanistic principles behind various antisense targets currently available for SMA therapy.

  4. Mechanistic principles of antisense targets for the treatment of Spinal Muscular Atrophy

    PubMed Central

    Singh, Natalia N.; Lee, Brian M.; DiDonato, Christine J.; Singh, Ravindra N.

    2015-01-01

    Spinal muscular atrophy (SMA) is a major neurodegenerative disorder of children and infants. SMA is primarily caused by low levels of SMN protein owing to deletions or mutations of the survival motor neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of the production of the functional SMN protein due to predominant skipping of exon 7. Several compounds, including antisense oligonucleotides (ASOs) that elevate SMN protein from SMN2 hold the promise for treatment. An ASO-based drug currently under phase 3 clinical trial employs intronic splicing silencer N1 (ISS-N1) as its target. Cumulative studies on the ISS-N1 reveal a wealth of information with significance to the overall therapeutic development for SMA. Here we summarize the mechanistic principles behind various antisense targets currently available for SMA therapy. PMID:26381381

  5. Kv1.1 channel antisense attenuates learning and modulation of dentate polysialylated NCAM.

    PubMed

    Gratacós, E; Ghelardini, C; Gherardini, L M; Galeotti, N; Murphy, K J; Bartolini, A; Regan, C M

    1998-08-24

    The distribution and modulation of neural cell adhesion molecule polysialylation state (NCAM PSA) and the consequence of antisense inactivation of the Kv1.1 potassium channel was investigated following avoidance learning in mice. PSA immunoreactivity was most notable on cells at the inner denate border and in cortical layer II. Task acquisition resulted in a significant 30% transient increase in the frequency of dentate polysialylated neurons at the 12 h post-training time. In contrast, animals pretreated with the Kv1.1 antisense oligonucleotide exhibited both attenuated recall avoidance latencies and polysialylated cell frequency. As Kv1.1 is enriched on the dendrites of these granule-like cells, the attenuated polysialylation response is considered secondary to NCAM-mediated events during their transient synapse production in the 6-8 h post-training period.

  6. Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.

    PubMed Central

    Maher, L J; Dolnick, B J

    1988-01-01

    Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage. Images PMID:2836793

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

    PubMed Central

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

    2016-01-01

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

  8. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligos

    PubMed Central

    Sztainberg, Yehezkel; Chen, Hong-mei; Swann, John W.; Hao, Shuang; Tang, Bin; Wu, Zhenyu; Tang, Jianrong; Wan, Ying-Wooi; Liu, Zhandong; Rigo, Frank; Zoghbi, Huda Y.

    2015-01-01

    Copy number variations have been frequently associated with developmental delay, intellectual disability, and autism spectrum disorders1. MECP2 duplication syndrome is one of the most common genomic rearrangements in males2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections, and early death3–5. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question, however, was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders6–8, including loss of MeCP2 in Rett syndrome9, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we hypothesized that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. Therefore, we first generated and characterized a conditional Mecp2-overexpressing mouse model and showed that correction of MeCP2 levels largely reversed the behavioral, molecular, and electrophysiological deficits. Next, we sought a translational strategy to reduce MeCP2 and turned to antisense oligonucleotides (ASOs). ASOs are small modified nucleic acids that can selectively hybridize with mRNA transcribed from a target gene and silence it10,11, and have been successfully used to correct deficits in different mouse models12–18. We found that ASO treatment induced a broad phenotypic rescue in adult symptomatic transgenic MECP2

  9. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides.

    PubMed

    Adachi, Shungo; Natsume, Tohru

    2015-01-01

    To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex.

  10. Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex

    PubMed Central

    Kralovicova, Jana; Lages, Ana; Patel, Alpa; Dhir, Ashish; Buratti, Emanuele; Searle, Mark; Vorechovsky, Igor

    2014-01-01

    Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression. PMID:24944197

  11. Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2014-06-01

    Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in

  12. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    PubMed

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  13. VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

    PubMed Central

    Gu, Zhong-Ping; Wang, Yun-Jie; Li, Jin-Ge; Zhou, Yong-An

    2002-01-01

    AIM: To investigate the effect of antisense RNA to vascular endothelial growth factor165 (VEGF165) on human esophageal squamous cell carcinoma cell line EC109 and the feasibility of gene therapy for esophageal carcinoma. METHODS: By using subclone technique, the full length of VEGF165 amino acid cDNA, which was cut from pGEM-3Zf(+), was cloned inversely into the eukaryotic expression vector pCEP4.The recombinant plasmid pCEP-AVEGF165 was transfected into EC109 cell with lipofectamine. After a stable transfection, dot blot, enzyme-linked immunosorbent assay (ELISA), laser confocal imaging system analysis, transmission electron microscopy and flow cytometry were performed to determine the biological characteristics of EC109 cell line before and after transfection in vitro and whether there was a reversion in the tumorigenic properties of the EC109 cell in vivo. RESULTS: The eukaryotic expression vector pCEP-AVEGF165 was successfully constructed and transfected into EC109 cells. The expression of VEGF165 was significantly decreased in the transfected cells while the biological characteristics of the cells were not influenced by the expression of antisense gene. The tumorigenic and angiogenic capabilities were greatly reduced in nude mice, as demonstrated by reduced tumor end volume (820 ± 112.5) mm3 vs (7930 ± 1035) mm3 and (7850 ± 950) mm3,P£¼0.01£½ and microvessel density(8.5 ± 1.2) mm-2 vs (44.3 ± 9.4) mm-2 and (46.4 ± 12.6) mm-2,P < 0.01) in comparison between experimental groups empty vector transfected group and control group. CONCLUSION: The angiogenesis and tumorigenicity of human esophageal squamous cell carcinoma were effectively inhibited by VEGF165 antisense RNA. Antisense RNA to VEGF165 can potentially be used as an adjuvant therapy for solid tumors. PMID:11833069

  14. Splicing of a non-coding antisense transcript controls LEF1 gene expression

    PubMed Central

    Beltran, Manuel; Aparicio-Prat, Estel; Mazzolini, Rocco; Millanes-Romero, Alba; Massó, Pere; Jenner, Richard G.; Díaz, Víctor M.; Peiró, Sandra; de Herreros, Antonio García

    2015-01-01

    In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms. PMID:25990740

  15. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

  16. Intrathecal PLC(β3) oligodeoxynucleotides antisense potentiates acute morphine efficacy and attenuates chronic morphine tolerance.

    PubMed

    Quanhong, Zhou; Ying, Xue; Moxi, Chen; Tao, Xu; Jing, Wang; Xin, Zhang; Li, Wang; Derong, Cui; Xiaoli, Zhang; Wei, Jiang

    2012-09-01

    Morphine is a mainstay for chronic pain treatment, but its efficacy has been hampered by physical tolerance. The underlying mechanism for chronic morphine induced tolerance is complicated and not well understood. PLC(β3) is regarded as an important factor in the morphine tolerance signal pathway. In this study, we determined intrathecal (i.t.) administration of an antisense oligodeoxynucleotide (ODN) of PLC(β3) could quicken the on-set antinociceptive efficacy of acute morphine treatment and prolong the maximum effect up to 4h. The antisense could also attenuate the development of morphine-induced tolerance and left shift the ED50 after 7 day of coadministration with morphine. These results probably were contributed by the PLC(β3) antisense ODN as they successfully knocked down protein expression levels and reduced activity of PLC(β3) in spinal cord in rats. The mismatch group had no such effects. The results confirmed the important involvement of PLC(β3) in both acute morphine efficacy and chronic morphine tolerance at spinal level in rats. This study may provide an idea for producing a novel adjuvant for morphine treatment.

  17. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. PMID:26534950

  18. Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

    PubMed Central

    Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2003-01-01

    Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432

  19. α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans.

    PubMed

    Lefimil, C; Jedlicki, E; Holmes, D S

    2014-03-01

    A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

  20. An antisense RNA that governs the expression kinetics of a multifunctional virulence gene

    PubMed Central

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

    Summary Genome-wide transcriptome analyses of several bacterial species have recently uncovered a hitherto unappreciated amount of antisense transcription. However, the physiological role, regulation and significance of such antisense transcripts are presently unclear. We now report the identification of a cis-encoded 1.2 kb long antisense RNA – termed AmgR – that is complementary to the mgtC portion of the mgtCBR polycistronic message from Salmonella enterica. The mgtCBR mRNA specifies the MgtC protein, which is necessary for survival within macrophages, virulence in mice and growth in low Mg2+; the Mg2+ transporter MgtB with no apparent role in virulence; and the membrane peptide MgtR mediating MgtC degradation. Expression of AmgR dimished both MgtC and MgtB protein levels in a process requiring RNase E but independent of RNase III, the RNA chaperone Hfq, and the regulatory peptide MgtR. Inactivation of the chromosomal amgR promoter increased MgtC and MgtB protein levels and enhanced Salmonella virulence. Surprisingly, AmgR transcription is governed by the regulatory protein PhoP, which also directs transcription of the sense mgtCBR mRNA. AmgR may function as a timing device that alters MgtC and MgtB levels after the onset of PhoP-inducing conditions. PMID:20398218

  1. Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

    PubMed Central

    Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J

    1994-01-01

    We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites. Images PMID:8058724

  2. Chemistry and biochemistry of 2',5'-oligoadenylate-based antisense strategy.

    PubMed

    Adah, S A; Bayly, S F; Cramer, H; Silverman, R H; Torrence, P F

    2001-08-01

    This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described. PMID:11472236

  3. PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

    PubMed

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

    2012-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

  4. Poly(propylacrylic acid) enhances cationic lipid mediated delivery of antisense oligonucleotides

    PubMed Central

    Lee, Li Kim; Williams, Charity L.; Devore, David; Roth, Charles M.

    2008-01-01

    The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved two- to threefold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides. PMID:16677032

  5. Inhibition of the synthesis of a cytochrome-c-oxidase subunit isoform by antisense RNA.

    PubMed

    Sandonà, D; Bisson, R

    1994-02-01

    To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity. PMID:8112318

  6. Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells.

    PubMed

    Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Understanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells. PMID:26986720

  7. A computational analysis of antisense off-targets in prokaryotic organisms.

    PubMed

    Worley-Morse, Thomas O; Gunsch, Claudia K

    2015-02-01

    The adoption of antisense gene silencing as a novel disinfectant for prokaryotic organisms is hindered by poor silencing efficiencies. Few studies have considered the effects of off-targets on silencing efficiencies, especially in prokaryotic organisms. In this computational study, a novel algorithm was developed that determined and sorted the number of off-targets as a function of alignment length in Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv. The mean number of off-targets per a single location was calculated to be 14.1 ± 13.3 and 36.1 ± 58.5 for the genomes of E. coli K-12 MG1655 and M. tuberculosis H37Rv, respectively. Furthermore, when the entire transcriptome was analyzed, it was found that there was no general gene location that could be targeted to minimize or maximize the number of off-targets. In an effort to determine the effects of off-targets on silencing efficiencies, previously published studies were used. Analyses with acpP, ino1, and marORAB revealed a statistically significant relationship between the number of short alignment length off-targets hybrids and the efficacy of the antisense gene silencing, suggesting that the minimization of off-targets may be beneficial for antisense gene silencing in prokaryotic organisms. PMID:25486012

  8. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

    PubMed

    Mitsudome, Yuya; Takahama, Mamiko; Hirose, Jun; Yoshida, Naoto

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for

  9. Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

    PubMed Central

    Jin, Hailing; Vacic, Vladimir; Girke, Thomas; Lonardi, Stefano; Zhu, Jian-Kang

    2008-01-01

    Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs). The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs) via the RNA interference (RNAi) pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1) inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2) generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP) and Massively Parallel Signature Sequencing (MPSS) small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64%) protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or mitochondrion

  10. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

    PubMed Central

    Nishimura, Yoshiki; Kikis, Elise A.; Zimmer, Sara L.; Komine, Yutaka; Stern, David B.

    2004-01-01

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5

  11. Oligonucleotides antisense to the interleukin 1 receptor mRNA block the effects of interleukin 1 in cultured murine and human fibroblasts and in mice.

    PubMed Central

    Burch, R M; Mahan, L C

    1991-01-01

    Phosphodiester and phosphorothioate oligodeoxynucleotides (18 mers) were constructed antisense to sequences of the recently cloned murine and human IL-1 receptors. Murine antisense oligonucleotides inhibited IL-1-stimulated PGE2 synthesis by murine fibroblasts in culture in a time (days) and concentration-dependent (3 microM-30 microM) fashion. Murine sense oligonucleotide and an oligonucleotide antisense to human IL-1 receptor were without effect. Moreover, murine antisense oligonucleotides did not affect tumor necrosis factor- or bradykinin-stimulated PGE2 synthesis by murine fibroblasts. Similarly, antisense oligonucleotides to the human, but not the murine, IL-1 receptor inhibited IL-1-stimulated PGE2 synthesis by cultured human fibroblasts. The attenuation of the cellular response to IL-1 caused by the antisense oligonucleotides correlated with a loss in cell surface receptors for IL-1, without any change in the number of bradykinin receptors on these cells. When antisense oligonucleotides were encapsulated in liposomes, they blocked completely the appearance of newly synthesized IL-1 receptors and IL-1-stimulated PGE2 synthesis. In mice, subcutaneous injection with an oligonucleotide antisense to the murine IL-1 receptor markedly inhibited the infiltration of neutrophils in response to subsequent injection of IL-1. These data suggest that antisense oligodeoxynucleotides may share a role in the design of antiinflammatory therapeutics. Images PMID:1833422

  12. [Multi-Target Recognition of Internal and External Defects of Potato by Semi-Transmission Hyperspectral Imaging and Manifold Learning Algorithm].

    PubMed

    Huang, Tao; Li, Xiao-yu; Jin, Rui; Ku, Jing; Xu, Sen-miao; Xu, Meng-ling; Wu, Zhen-zhong; Kong, De-guo

    2015-04-01

    The present paper put forward a non-destructive detection method which combines semi-transmission hyperspectral imaging technology with manifold learning dimension reduction algorithm and least squares support vector machine (LSSVM) to recognize internal and external defects in potatoes simultaneously. Three hundred fifteen potatoes were bought in farmers market as research object, and semi-transmission hyperspectral image acquisition system was constructed to acquire the hyperspectral images of normal external defects (bud and green rind) and internal defect (hollow heart) potatoes. In order to conform to the actual production, defect part is randomly put right, side and back to the acquisition probe when the hyperspectral images of external defects potatoes are acquired. The average spectrums (390-1,040 nm) were extracted from the region of interests for spectral preprocessing. Then three kinds of manifold learning algorithm were respectively utilized to reduce the dimension of spectrum data, including supervised locally linear embedding (SLLE), locally linear embedding (LLE) and isometric mapping (ISOMAP), the low-dimensional data gotten by manifold learning algorithms is used as model input, Error Correcting Output Code (ECOC) and LSSVM were combined to develop the multi-target classification model. By comparing and analyzing results of the three models, we concluded that SLLE is the optimal manifold learning dimension reduction algorithm, and the SLLE-LSSVM model is determined to get the best recognition rate for recognizing internal and external defects potatoes. For test set data, the single recognition rate of normal, bud, green rind and hollow heart potato reached 96.83%, 86.96%, 86.96% and 95% respectively, and he hybrid recognition rate was 93.02%. The results indicate that combining the semi-transmission hyperspectral imaging technology with SLLE-LSSVM is a feasible qualitative analytical method which can simultaneously recognize the internal and

  13. Phase II trial of pazopanib (GW786034), an oral multi-targeted angiogenesis inhibitor, for adults with recurrent glioblastoma (North American Brain Tumor Consortium Study 06-02)

    PubMed Central

    Iwamoto, Fabio M.; Lamborn, Kathleen R.; Robins, H. Ian; Mehta, Minesh P.; Chang, Susan M.; Butowski, Nicholas A.; DeAngelis, Lisa M.; Abrey, Lauren E.; Zhang, Wei-Ting; Prados, Michael D.; Fine, Howard A.

    2010-01-01

    The objective of this phase II single-arm study was to evaluate the efficacy and safety of pazopanib, a multi-targeted tyrosine kinase inhibitor, against vascular endothelial growth factor receptor (VEGFR)-1, -2, and -3, platelet-derived growth factor receptor-α and -β, and c-Kit, in recurrent glioblastoma. Patients with ≤2 relapses and no prior anti-VEGF/VEGFR therapy were treated with pazopanib 800 mg daily on 4-week cycles without planned interruptions. Brain magnetic resonance imaging and clinical reassessment were made every 8 weeks. The primary endpoint was efficacy as measured by 6-month progression-free survival (PFS6). Thirty-five GBM patients with a median age of 53 years and median Karnofsky performance scale of 90 were accrued. Grade 3/4 toxicities included leukopenia (n = 1), lymphopenia (n = 2), thrombocytopenia (n = 1), ALT elevation (n = 3), AST elevation (n = 1), CNS hemorrhage (n = 1), fatigue (n = 1), and thrombotic/embolic events (n = 3); 8 patients required dose reduction. Two patients had a partial radiographic response by standard bidimensional measurements, whereas 9 patients (6 at the 8-week point and 3 only within the first month of treatment) had decreased contrast enhancement, vasogenic edema, and mass effect but <50% reduction in tumor. The median PFS was 12 weeks (95% confidence interval [CI]: 8–14 weeks) and only 1 patient had a PFS time ≥6 months (PFS6 = 3%). Thirty patients (86%) had died and median survival was 35 weeks (95% CI: 24–47 weeks). Pazopanib was reasonably well tolerated with a spectrum of toxicities similar to other anti-VEGF/VEGFR agents. Single-agent pazopanib did not prolong PFS in this patient population but showed in situ biological activity as demonstrated by radiographic responses. ClinicalTrials.gov identifier: NCT00459381. PMID:20200024

  14. A Network Pharmacology Study of Chinese Medicine QiShenYiQi to Reveal Its Underlying Multi-Compound, Multi-Target, Multi-Pathway Mode of Action

    PubMed Central

    Li, Xiang; Wu, Leihong; Liu, Wei; Jin, Yecheng; Chen, Qian; Wang, Linli; Fan, Xiaohui; Li, Zheng; Cheng, Yiyu

    2014-01-01

    Chinese medicine is a complex system guided by traditional Chinese medicine (TCM) theories, which has proven to be especially effective in treating chronic and complex diseases. However, the underlying modes of action (MOA) are not always systematically investigated. Herein, a systematic study was designed to elucidate the multi-compound, multi-target and multi-pathway MOA of a Chinese medicine, QiShenYiQi (QSYQ), on myocardial infarction. QSYQ is composed of Astragalus membranaceus (Huangqi), Salvia miltiorrhiza (Danshen), Panax notoginseng (Sanqi), and Dalbergia odorifera (Jiangxiang). Male Sprague Dawley rat model of myocardial infarction were administered QSYQ intragastrically for 7 days while the control group was not treated. The differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with QSYQ, followed by constructing a cardiovascular disease (CVD)-related multilevel compound-target-pathway network connecting main compounds to those DEGs supported by literature evidences and the pathways that are functionally enriched in ArrayTrack. 55 potential targets of QSYQ were identified, of which 14 were confirmed in CVD-related literatures with experimental supporting evidences. Furthermore, three sesquiterpene components of QSYQ, Trans-nerolidol, (3S,6S,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol and (3S,6R,7R)-3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol from Dalbergia odorifera T. Chen, were validated experimentally in this study. Their anti-inflammatory effects and potential targets including extracellular signal-regulated kinase-1/2, peroxisome proliferator-activated receptor-gamma and heme oxygenase-1 were identified. Finally, through a three-level compound-target-pathway network with experimental analysis, our study depicts a complex MOA of QSYQ on myocardial infarction. PMID:24817581

  15. Anti-myeloma activity of a multi targeted kinase inhibitor, AT9283, via potent Aurora Kinase and STAT3 inhibition either alone or in combination with lenalidomide

    PubMed Central

    Santo, Loredana; Hideshima, Teru; Cirstea, Diana; Bandi, Madhavi; Nelson, Erik A.; Gorgun, Gullu; Rodig, Scott; Vallet, Sonia; Pozzi, Samantha; Patel, Kishan; Unitt, Christine; Squires, Matt; Hu, Yiguo; Chauhan, Dharminder; Mahindra, Anuj; Munshi, Nikhil C.; Anderson, Kenneth C.; Raje, Noopur

    2014-01-01

    Purpose Aurora Kinases, whose expression is linked to genetic instability and cellular proliferation, are under investigation as novel therapeutic targets in multiple myeloma (MM). Here, we investigated the preclinical activity of a small molecule–multi-targeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Janus Kinase 2/3. Experimental design We evaluated the in vitro anti myeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a Xenograft mouse model of human MM. Results Our data demonstrated AT9283 induced cell growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both AURKA and AURKB inhibition, e.g increase of cells with polyploid DNA content, decrease in phospho-Histone H3, and decrease of phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, AURKA or AURKB showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies demonstrated decreased MM cell growth and prolonged survival in AT9283-treated mice compared to controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells (BMSCs). Enhanced cytotoxicity was associated with increased inhibition of pSTAT3 and pERK. Conclusions Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination in patients with MM. PMID:21430070

  16. New insights into pharmacological profile of LASSBio-579, a multi-target N-phenylpiperazine derivative active on animal models of schizophrenia.

    PubMed

    Neves, Gilda; Antonio, Camila B; Betti, Andresa H; Pranke, Mariana A; Fraga, Carlos A M; Barreiro, Eliezer J; Noël, François; Rates, Stela M K

    2013-01-15

    Previous behavioral and receptor binding studies on N-phenylpiperazine derivatives by our group indicated that LASSBio-579, LASSBio-580 and LASSBio-581 could be potential antipsychotic lead compounds. The present study identified LASSBio-579 as the most promising among the three compounds, since it was the only one that inhibited apomorphine-induced climbing (5 mg/kg p.o.) and apomorphine-induced hypothermia (15 mg/kg p.o.). Furthermore, LASSBio-579 (0.5 mg/kg p.o.) was effective in the ketamine-induced hyperlocomotion test and prevented the prepulse inhibition deficits induced by apomorphine, DOI and ketamine with different potencies (1 mg/kg, 0.5 mg/kg and 5 mg/kg p.o., respectively). LASSBio-579 also induced a motor impairment, catalepsy and a mild sedative effect but only at doses 3-120 times higher than those with antipsychotic-like effects. In addition, LASSBio-579 (0.5 and 1 mg/kg p.o.) reversed the catalepsy induced by WAY 100,635, corroborating its action on both dopaminergic and serotonergic neurotransmission and pointing to the contribution of 5-HT(1A) receptor activation to its pharmacological profile. Moreover, co-administration of sub-effective doses of LASSBio-579 with sub-effective doses of clozapine or haloperidol prevented the apomorphine-induced climbing without induction of catalepsy. In summary, our results characterize LASSBio-579 as a multi-target ligand active in pharmacological animal models of schizophrenia, confirming that this compound could be included in development programs aiming at a new drug for treating schizophrenia.

  17. Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes.

    PubMed

    Lu, Xueguang; Jia, Fei; Tan, Xuyu; Wang, Dali; Cao, Xueyan; Zheng, Jiamin; Zhang, Ke

    2016-07-27

    Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics. PMID:27420413

  18. Antisense-oligonucleotide mediated exon skipping in activin-receptor-like kinase 2: inhibiting the receptor that is overactive in fibrodysplasia ossificans progressiva.

    PubMed

    Shi, Songting; Cai, Jie; de Gorter, David J J; Sanchez-Duffhues, Gonzalo; Kemaladewi, Dwi U; Hoogaars, Willem M H; Aartsma-Rus, Annemieke; 't Hoen, Peter A C; ten Dijke, Peter

    2013-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.

  19. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

    PubMed Central

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-01-01

    Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

  20. Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

    PubMed Central

    Zwicky, Roman; Müntener, Kathrin; Goldring, Mary B; Baici, Antonio

    2002-01-01

    Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies. PMID:12086583

  1. Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

    PubMed

    Boopathi, P A; Subudhi, Amit Kumar; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Chand, Bipin; Mugasimangalam, Raja C; Kochar, Sanjay K; Sirohi, Parmendra; Kochar, Dhanpat K; Das, Ashis

    2013-12-01

    Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

  2. CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences

    PubMed Central

    Prykhozhij, Sergey V.; Rajan, Vinothkumar; Gaston, Daniel; Berman, Jason N.

    2015-01-01

    Genome engineering has been revolutionized by the discovery of clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar sequences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we developed the CRISPR MultiTargeter software. Similar DNA sequences in question are duplicated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas9 nickase targeting, as well as common and unique sgRNA target searches in 1) a set of input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. We demonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in 71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40% of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpful because it will facilitate functional genomic studies of transcript isoforms. Similarly, its application to duplicated genes may simplify multi-gene mutational targeting experiments. Overall, this program provides a unique interface that will enhance use of CRISPR/Cas technology. PMID:25742428

  3. Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene.

    PubMed

    Sohrab, Sayed Sartaj; Kamal, Mohammad A; Ilah, Abdul; Husen, Azamal; Bhattacharya, P S; Rana, D

    2016-05-01

    Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV. PMID:27081361

  4. Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene

    PubMed Central

    Sohrab, Sayed Sartaj; Kamal, Mohammad A.; Ilah, Abdul; Husen, Azamal; Bhattacharya, P.S.; Rana, D.

    2014-01-01

    Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV. PMID:27081361

  5. Antisense molecular beacon strategy for in situ visualization of snRNA and fibrillarin protein interaction in Giardia lamblia.

    PubMed

    Ganguly, Sandipan; Ghosh, Srikanta; Chattopadhyay, Dhrubajyoti; Das, Pradeep

    2004-05-01

    Use of confocal microscopy has provided many recent developments in the study of functional aspects, especially localization and distribution of proteins, DNA and RNA within the cells. In the present investigation, we have applied for the first time, antisense molecular beacon based Fluorescence Resonance Energy Transfer (FRET) and Flow Cytometric Energy Transfer (FCET) techniques to demonstrate binding and co-localization of fibrillarin protein with small nuclear RNA (snRNA) to form ribonucleoprotein particle (RNPP) complex in Giardia lamblia. It has been observed by FRET and FCET that energy transfer occurs from fluorescence tagged fibrillarin to snRNA antisense molecular beacon confirming the clear physical interaction between them during RNPP complex formation. This is the first demonstration of in situ detection of RNA-protein complex formation by antisense molecular beacon based FRET and FCET in Giardia lamblia.

  6. The effects of antisense to Gialpha2 on opioid agonist potency and Gialpha2 protein and mRNA abundance in the mouse.

    PubMed

    Shen, J; Shah, S; Hsu, H; Yoburn, B C

    1998-08-31

    In this study, mice received a single intracerebroventricular (i.c.v. ) injection of an antisense oligodeoxynucleotide (ODN) directed towards the mRNA of Gialpha2. Controls received a saline or a nonsense ODN injection. The subsequent effects on protein levels and mRNA of Gialpha2 were determined in mouse striatum, as well as, the effect on opioid ([d-Ala2, d-Leu5]-enkephalin; DADLE) inhibition of cyclic AMP (cAMP) formation in striatum and morphine analgesic potency. At 48 h after treatment, maximal inhibition (Emax) of cAMP formation was significantly reduced for the antisense group compared to controls. Antisense ODN treatment only changed the Emax and did not significantly alter the IC50s of the dose-effect curves for inhibition of cAMP formation. Antisense ODN, but not nonsense ODN, significantly reduced morphine's analgesic potency by >2-fold, 48 h following treatment. Using a quantitative immunoblotting procedure, antisense treatment was shown to decrease striatal Gialpha2 protein 48 h after antisense injection, while there were no changes in protein levels at 2, 12 and 24 h. In contrast, no changes in Gialpha2 mRNA in mouse striatum were noted at any time after antisense treatment. Taken together, these data suggest that Gialpha2 mediates opioid-induced analgesia and opioid inhibition of cAMP production in the mouse. These data also suggest that antisense reduces target protein by a mechanism independent of changes in mRNA abundance.

  7. Antisense-mediated depletion of GMPase gene expression in tobacco decreases plant tolerance to temperature stresses and alters plant development.

    PubMed

    Wang, Hua-Sen; Zhu, Zhu-Jun; Feng, Zhen; Zhang, Shi-Gang; Yu, Chao

    2012-12-01

    In our previous work [1] we investigated the role of tomato GDP-mannose pyrophosphorylase (EC 2.7.7.22) in plants by overexpressing its gene in tobacco leaves and showed its function in AsA metabolism and detoxification of reactive oxygen species under temperature stresses. In this study, we use the antisense technique to block the endogenous GMPase gene expression in tobacco in order to further investigate its function. Northern and western blot analysis confirmed that the expression of endogenous tobacco GMPase mRNA and protein was inhibited by this antisense expression. Consequently, the activity of GMPase and the content of AsA in the leaves of antisense transgenic plants were markedly decreased. This was also the case for the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves of the transgenic plants. On the contrary, the contents of H(2)O(2) and O(2) (-•) were increased. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) also declined in the leaves of antisense plants. Under high or low temperature stresses, the seed germination rate of the antisense transgenic plants was significantly decreased in comparison with that of the wild-type tobacco. Interestingly, the antisense plants had smaller leaves and an earlier onset of flowering. In conclusion, the depletion of GMPase decreased the content of AsA, resulting in the plants susceptible to the oxidative damage caused by temperature stresses and subjected to developmental alternations.

  8. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    PubMed

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.

  9. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    PubMed

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae. PMID:23728780

  10. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

    PubMed Central

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E.

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration. PMID:26366578

  11. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Migawa, Michael T; Vickers, Timothy A; Crooke, Stanley T

    2016-08-01

    Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression or to modify RNA splicing, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30-150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an ∼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility. PMID:27398791

  12. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

    PubMed Central

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    ABSTRACT Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation. PMID:26580233

  13. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic

    PubMed Central

    Cassan, Elodie; Arigon-Chifolleau, Anne-Muriel; Mesnard, Jean-Michel; Gross, Antoine; Gascuel, Olivier

    2016-01-01

    Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame −2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp. Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins—an expected outcome with overlaps in frame −2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic. PMID:27681623

  14. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  15. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  16. Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    PubMed Central

    Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M.; Fei, Jingyi; Ha, Taekjip; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2016-01-01

    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing. PMID:26826711

  17. Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense Rab11 GTPase gene.

    PubMed

    Lu, C; Zainal, Z; Tucker, G A; Lycett, G W

    2001-08-01

    A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall-modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a "mutant phenotype." Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.

  18. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    PubMed

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

  19. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  20. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

    PubMed Central

    Boiziau, C; Kurfurst, R; Cazenave, C; Roig, V; Thuong, N T; Toulmé, J J

    1991-01-01

    We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H. Images PMID:1850511

  1. Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants.

    PubMed

    Zeh, M; Casazza, A P; Kreft, O; Roessner, U; Bieberich, K; Willmitzer, L; Hoefgen, R; Hesse, H

    2001-11-01

    Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality. PMID:11706163

  2. Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

    SciTech Connect

    Anai, Satoshi; Brown, Bob D.; Nakamura, Kogenta; Goodison, Steve; Hirao, Yoshihiko; Rosser, Charles J. . E-mail: charles.rosser@urology.ufl.edu

    2007-07-15

    Purpose: To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. Methods and Materials: PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. Results: The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. Conclusions: These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications.

  3. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    1999-09-28

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.

  4. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

    PubMed

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

  5. Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

    PubMed

    Glas, Julia; Dümcke, Sebastian; Zacher, Benedikt; Poron, Don; Gagneur, Julien; Tresch, Achim

    2016-03-18

    Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor. PMID:26578558

  6. Lipid-Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α.

    PubMed

    Li, Hong; Quan, Jishan; Zhang, Mengzi; Yung, Bryant C; Cheng, Xinwei; Liu, Yang; Lee, Young B; Ahn, Chang-Ho; Kim, Deog Joong; Lee, Robert J

    2016-07-01

    Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 μM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

  7. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    PubMed

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  8. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    PubMed

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  9. Additive effect of mPer1 and mPer2 antisense oligonucleotides on light-induced phase shift.

    PubMed

    Wakamatsu, H; Takahashi, S; Moriya, T; Inouye, S T; Okamura, H; Akiyama, M; Shibata, S

    2001-01-22

    It is well known that light induces both mPer1 and mPer2 mRNA in the suprachiasmatic nucleus. We have reported that mPer1 antisense oligonucleotides (ODNs) inhibited the light-induced phase delays of mouse locomotor rhythm. In this study, we asked whether both or either mPer1 or mPer2 expression is necessary to induce the phase shift. We examined the effects of inhibition of mRNA expression on light-induced phase delays of mouse circadian behavior rhythm. Light-induced phase delays were moderately attenuated by microinjection of mPer1 or mPer2 antisense ODN, but not by mPer3 antisense or mPer1, mPer2 scrambled ODNs, whereas following simultaneous injection of both mPer1 and mPer2 antisense ODNs they disappeared. The present results suggest that acute induction of mPer1 and mPer2 gene play an additive effect on photic entrainment. PMID:11201072

  10. Dataset of natural antisense transcripts in P. vivax clinical isolates derived using custom designed strand-specific microarray

    PubMed Central

    Boopathi, P.A.; Subudhi, Amit Kumar; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Chand, Bipin; Mugasimangalam, Raja C.; Kochar, Sanjay K.; Sirohi, Parmendra; Kochar, Dhanpat K.; Das, Ashis

    2014-01-01

    Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013. PMID:26484095

  11. Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

    PubMed

    Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

    2013-07-01

    Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

  12. Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.

    PubMed Central

    Khillan, J S; Li, S W; Prockop, D J

    1994-01-01

    Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes. Images PMID:8022775

  13. Topical gene silencing by iontophoretic delivery of an antisense oligonucleotide-dendrimer nanocomplex: the proof of concept in a skin cancer mouse model

    NASA Astrophysics Data System (ADS)

    Venuganti, , Venkata Vamsi K.; Saraswathy, Manju; Dwivedi, Chandradhar; Kaushik, Radhey S.; Perumal, Omathanu P.

    2015-02-01

    The study was aimed at investigating the feasibility of using a poly (amidoamine) (PAMAM) dendrimer as a carrier for topical iontophoretic delivery of an antisense oligonucleotide (ASO). Bcl-2, an anti-apoptotic protein implicated in skin cancer, was used as the model target protein to demonstrate the topical gene silencing approach. Confocal laser scanning microscopy studies demonstrated that the iontophoretically delivered ASO-dendrimer complex can reach the viable epidermis in porcine skin. In contrast, passively delivered free or dendrimer complexed ASO was mainly localized to the stratum corneum. The cell uptake of ASO was significantly enhanced by the dendrimer complex and the complex suppressed Bcl-2 levels in the cell. In the skin cancer mouse model, the iontophoretically delivered ASO-dendrimer complex reduced the tumor volume by 45% and was consistent with the reduction in Bcl-2 protein levels. The iontophoretically delivered ASO-dendrimer complex caused significant apoptosis in skin tumor. Overall, the findings from this study demonstrate that dendrimers are promising nanocarriers for developing topical gene silencing approaches for skin diseases.The study was aimed at investigating the feasibility of using a poly (amidoamine) (PAMAM) dendrimer as a carrier for topical iontophoretic delivery of an antisense oligonucleotide (ASO). Bcl-2, an anti-apoptotic protein implicated in skin cancer, was used as the model target protein to demonstrate the topical gene silencing approach. Confocal laser scanning microscopy studies demonstrated that the iontophoretically delivered ASO-dendrimer complex can reach the viable epidermis in porcine skin. In contrast, passively delivered free or dendrimer complexed ASO was mainly localized to the stratum corneum. The cell uptake of ASO was significantly enhanced by the dendrimer complex and the complex suppressed Bcl-2 levels in the cell. In the skin cancer mouse model, the iontophoretically delivered ASO-dendrimer complex

  14. RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts.

    PubMed

    Peters, Nick T; Rohrbach, Justin A; Zalewski, Brian A; Byrkett, Colleen M; Vaughn, Jack C

    2003-06-01

    We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

  15. Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

    PubMed

    Smith, Alvin W; Matson, David O; Stein, David A; Skilling, Douglas E; Kroeker, Andrew D; Berke, Tamas; Iversen, Patrick L

    2002-04-01

    The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases. PMID:12044040

  16. Targeting Long Noncoding RNA with Antisense Oligonucleotide Technology as Cancer Therapeutics.

    PubMed

    Zhou, Tianyuan; Kim, Youngsoo; MacLeod, A Robert

    2016-01-01

    Recent annotation of the human transcriptome revealed that only 2 % of the genome encodes proteins while the majority of human genome is transcribed into noncoding RNAs. Although we are just beginning to understand the diverse roles long noncoding RNAs (lncRNAs) play in molecular and cellular processes, they have potentially important roles in human development and pathophysiology. However, targeting of RNA by traditional structure-based design of small molecule inhibitors has been difficult, due to a lack of understanding of the dynamic tertiary structures most RNA molecules adopt. Antisense oligonucleotides (ASOs) are capable of targeting specific genes or transcripts directly through Watson-Crick base pairing and thus can be designed based on sequence information alone. These agents have made possible specific targeting of "non-druggable targets" including RNA molecules. Here we describe how ASOs can be applied in preclinical studies to reduce levels of lncRNAs of interest.

  17. Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

    PubMed

    Onishi, Reina; Watanabe, Ayahisa; Nakajima, Mado; Sekiguchi, Mitsuaki; Kugimiya, Akira; Kinouchi, Hiroki; Nihashi, Yoichiro; Kamimori, Hiroshi

    2015-01-01

    In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

  18. Expansion of antisense lncRNA transcriptomes since the loss of RNAi

    PubMed Central

    Alcid, Eric A.; Tsukiyama, Toshio

    2016-01-01

    Antisense long noncoding RNAs (ASlncRNAs) have been implicated in regulating gene expression in response to physiological cues. However, little is known about ASlncRNA evolutionary dynamics, and what underlies the evolution of their expression. Here, using budding yeast species Saccharomyces and Naumovozyma as models, we show that ASlncRNA repertoires have expanded since the loss of RNAi, in terms of their expression levels, their lengths, and their degree of overlap with coding genes. Furthermore, we show RNAi is inhibitory to ASlncRNA transcriptomes, and that elevation of ASlncRNAs in the presence of RNAi is deleterious to Naumovozyma castellii, a natural host of RNAi. Together, our work suggests that the loss of RNAi had a substantial impact on the genome-wide increase in expression of ASlncRNAs across budding yeast evolution. PMID:27018804

  19. Axonal trafficking of an antisense RNA transcribed from a pseudogene is regulated by classical conditioning

    PubMed Central

    Korneev, Sergei A.; Kemenes, Ildiko; Bettini, Natalia L.; Kemenes, George; Staras, Kevin; Benjamin, Paul R.; O'Shea, Michael

    2013-01-01

    Natural antisense transcripts (NATs) are endogenous RNA molecules that are complementary to known RNA transcripts. The functional significance of NATs is poorly understood, but their prevalence in the CNS suggests a role in brain function. Here we investigated a long NAT (antiNOS-2 RNA) associated with the regulation of nitric oxide (NO) production in the CNS of Lymnaea, an established model for molecular analysis of learning and memory. We show the antiNOS-2 RNA is axonally trafficked and demonstrate that this is regulated by classical conditioning. Critically, a single conditioning trial changes the amount of antiNOS-2 RNA transported along the axon. This occurs within the critical time window when neurotransmitter NO is required for memory formation. Our data suggest a role for the antiNOS-2 RNA in establishing memories through the regulation of NO signaling at the synapse. PMID:23293742

  20. Efficient Synthesis and Biological Evaluation of 5'-GalNAc Conjugated Antisense Oligonucleotides.

    PubMed

    Østergaard, Michael E; Yu, Jinghua; Kinberger, Garth A; Wan, W Brad; Migawa, Michael T; Vasquez, Guillermo; Schmidt, Karsten; Gaus, Hans J; Murray, Heather M; Low, Audrey; Swayze, Eric E; Prakash, Thazha P; Seth, Punit P

    2015-08-19

    Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.

  1. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    PubMed Central

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  2. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    PubMed

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  3. Antisense inhibition of the photosystem I antenna protein Lhca4 in Arabidopsis thaliana.

    PubMed Central

    Zhang, H; Goodman, H M; Jansson, S

    1997-01-01

    The function of Lhca4, a gene encoding the photosystem 1 type IV chlorophyll a/b-binding protein complex in Arabidopsis, was investigated using antisense technology. Lhca4 protein was reduced in a number of mutant lines and abolished in one. The inhibition of protein was not correlated with the inhibition of mRNA. No depletion of Lhca1 was observed, but the low-temperature fluorescence emission spectrum was drastically altered in the mutants. The emission maximum was blue-shifted by 6 nm, showing that chlorophyll molecules bound to Lhca4 are responsible for most of the long-wavelength fluorescence emission. Some mutants also showed an unexplainable delay in flowering time and an increase in seed weight. PMID:9414561

  4. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    NASA Astrophysics Data System (ADS)

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  5. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    PubMed

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes. PMID:26847692

  6. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    PubMed

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj

    2008-08-01

    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  7. Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

    PubMed

    Hotta, Carlos Takeshi; Nishiyama, Milton Yutaka; Souza, Glaucia Mendes

    2013-01-01

    Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.

  8. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    PubMed

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  9. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  10. An Antisense RNA-mediated Mechanism Eliminates a Meiosis-specific Copper-regulated Transcript in Mitotic Cells*

    PubMed Central

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-01-01

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1+ gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1+ sense transcript, inhibiting cum1+ expression in vegetative cells. Inhibition of cum1+ transcription was not at the level of its initiation because fusion of the cum1+ promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1+-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1+ led to the exclusive production of a shorter iss1+ mRNA isoform, thereby allowing accumulation of cum1+ sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1+ antisense mRNA could pair with cum1+ sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1+ sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells. PMID:26229103

  11. An antisense RNA-mediated mechanism eliminates a meiosis-specific copper-regulated transcript in mitotic cells.

    PubMed

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-09-11

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

  12. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

    PubMed Central

    Johnson, Barbara W.; Olson, Ken E.; Allen-Miura, Tanya; Rayms-Keller, Alfredo; Carlson, Jonathan O.; Coates, Craig J.; Jasinskiene, Nijole; James, Anthony A.; Beaty, Barry J.; Higgs, Stephen

    1999-01-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  13. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

    PubMed

    Johnson, B W; Olson, K E; Allen-Miura, T; Rayms-Keller, A; Carlson, J O; Coates, C J; Jasinskiene, N; James, A A; Beaty, B J; Higgs, S

    1999-11-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  14. Inhibition of breast cancer growth in vivo by antiangiogenesis gene therapy with adenovirus-mediated antisense-VEGF

    PubMed Central

    Im, S-A; Kim, J-S; Gomez-Manzano, C; Fueyo, J; Liu, T-J; Cho, M-S; Seong, C-M; Lee, S N; Hong, Y-K; Yung, W K A

    2001-01-01

    Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-αVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-αVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-αVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF 165 cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-αVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336478

  15. Oral delivery of nanoparticles containing anticancer SN38 and hSET1 antisense for dual therapy of colon cancer.

    PubMed

    Dinarvand, M; Kiani, M; Mirzazadeh, F; Esmaeili, A; Mirzaie, Z; Soleimani, M; Dinarvand, R; Atyabi, F

    2015-01-01

    An oral delivery system intended for treatment of colon cancer in HT29 cancerous cells was investigated by encapsulating hSET1 antisense and SN38 anticancer in nanoparticles based on cysteine trimethyl chitosan (cysTMC) and carboxymethyl dextran (CMD). Studies have shown hSET1 as the main type of histone methyltransferase (HMT) complex, is significantly overexpressed in malignant cells. In this study, hSET1 antisense was employed to inhibit gene expression. Additionally, SN38 was incorporated into nanoparticles to enhance the efficiency of the system by inhibition of topoisomerase 1. CysTMC was synthetized and characterized by (1)H NMR and FTIR. Nanoparticles were prepared through complexation of CMD and cysTMC. Particle size and surface charge was 100-150 nm and 17-21 mV respectively with drug content of around 2.6%. Gel electrophoresis assay proved the stability of antisense in simulated gastric and intestinal fluids. Nanoparticles showed high mucoadhesion and glutathione responsive release. Cellular uptake was observed by confocal microscopy and quantified by flow cytometry. Cytotoxicity of NPs was assessed using MTT assay. Results showed hSET1/SN38 nanoparticles had significantly higher cytotoxicity against HT29 cells compared with nanoparticles containing SN38, free SN38 or naked hSET1. Therefore, present system could be considered an effective combination therapy of highly hydrophobic SN38 and hSET1.

  16. Antisense mRNA for NPY-Y1 receptor in the medial preoptic area increases prolactin secretion.

    PubMed

    Silveira, N A; Franci, C R

    1999-09-01

    We investigated the participation of neuropeptide Y-Y1 receptors within the medial preoptic area in luteinizing hormone, follicle-stimulating hormone and prolactin release. Four bilateral microinjections of sense (control) or antisense 18-base oligonucleotides of messenger ribonucleic acid (mRNA) (250 ng) corresponding to the NH2-terminus of the neuropeptide Y1 receptor were performed at 12-h intervals for two days into the medial preoptic area of ovariectomized Wistar rats (N = 16), weighing 180 to 200 g, treated with estrogen (50 microg) and progesterone (25 mg) two days before the experiments between 8.00 and 10:00 a.m. Blockade of Y1 receptor synthesis in the medial preoptic area by the antisense mRNA did not change plasma luteinizing hormone or follicle-stimulating hormone but did increase prolactin from 19.6 +/- 5.9 ng/ml in the sense group to 52.9 +/- 9.6 ng/ml in the antisense group. The plasma hormones were measured by radioimmunoassay and the values are reported as mean +/- SEM. These data suggest that endogenous neuropeptide Y in the medial preoptic area has an inhibitory action on prolactin secretion through Y1 receptors.

  17. Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

    PubMed Central

    Sioud, M

    1997-01-01

    The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs. PMID:9016562

  18. Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

    PubMed Central

    Pierce, M L; Ruffner, D E

    1998-01-01

    Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

  19. Improvement in the quality of seed storage protein by transformation of Brassica napus with an antisense gene for cruciferin.

    PubMed

    Kohno-Murase, J; Murase, M; Ichikawa, H; Imamura, J

    1995-09-01

    The levels of certain essential amino acids, in particular cysteine, lysine and methionine, in the seed storage protein of a commercial spring variety of rape, Brassica napus, have been increased by the introduction of an antisense gene for cruciferin, which is the most abundant storage protein in rapeseed. The antisense construct contained part of the cruA gene in an inverted orientation, and the gene was driven by the 5' flanking region of the gene for napin such that antisense RNA was expressed in a seed-specific manner. The construct was introduced by Agrobacterium-mediated gene transfer. In self-pollinated seeds (T1 seeds) of transgenic plants there was a reduction in the levels of the α1β1 and α2/3β2/3 subunits of cruciferin, whereas the level of the α4β4 subunit was unchanged. The total protein and lipid contents of transgenic seeds did not differ significantly from that of normal seeds. Seeds with reduced amounts of cruciferin accumulated higher amounts of napin than non-transformed seeds, but the level of oleosin was unaffected. Amino-acid analysis of the seed storage protein revealed that T1 seeds with reduced amounts of cruciferin contained higher relative levels of three essential amino acids, namely, lysine, methionine and cysteine, with increases of 10%, 8% and 32% over the respective levels in non-transgenic seeds (B. napus cv Westar). PMID:24169890

  20. Hormone-dependent expression of a steroidogenic acute regulatory protein natural antisense transcript in MA-10 mouse tumor Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3'-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5' and 3' RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.

  1. Oral delivery of nanoparticles containing anticancer SN38 and hSET1 antisense for dual therapy of colon cancer.

    PubMed

    Dinarvand, M; Kiani, M; Mirzazadeh, F; Esmaeili, A; Mirzaie, Z; Soleimani, M; Dinarvand, R; Atyabi, F

    2015-01-01

    An oral delivery system intended for treatment of colon cancer in HT29 cancerous cells was investigated by encapsulating hSET1 antisense and SN38 anticancer in nanoparticles based on cysteine trimethyl chitosan (cysTMC) and carboxymethyl dextran (CMD). Studies have shown hSET1 as the main type of histone methyltransferase (HMT) complex, is significantly overexpressed in malignant cells. In this study, hSET1 antisense was employed to inhibit gene expression. Additionally, SN38 was incorporated into nanoparticles to enhance the efficiency of the system by inhibition of topoisomerase 1. CysTMC was synthetized and characterized by (1)H NMR and FTIR. Nanoparticles were prepared through complexation of CMD and cysTMC. Particle size and surface charge was 100-150 nm and 17-21 mV respectively with drug content of around 2.6%. Gel electrophoresis assay proved the stability of antisense in simulated gastric and intestinal fluids. Nanoparticles showed high mucoadhesion and glutathione responsive release. Cellular uptake was observed by confocal microscopy and quantified by flow cytometry. Cytotoxicity of NPs was assessed using MTT assay. Results showed hSET1/SN38 nanoparticles had significantly higher cytotoxicity against HT29 cells compared with nanoparticles containing SN38, free SN38 or naked hSET1. Therefore, present system could be considered an effective combination therapy of highly hydrophobic SN38 and hSET1. PMID:25858880

  2. Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes.

    PubMed Central

    Hagenbuch, B; Scharschmidt, B F; Meier, P J

    1996-01-01

    A Na(+)-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na(+)-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na(+)-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na(+)-dependent taurocholate uptake, but it blocked Na(+)-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na(+)-dependent and Na(+)-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver. PMID:8670169

  3. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    PubMed Central

    Falzarano, Maria Sofia; Passarelli, Chiara

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782

  4. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    PubMed

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  5. Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.

    PubMed

    Lok, Chun-Nam; Viazovkina, Ekaterina; Min, Kyung-Lyum; Nagy, Eva; Wilds, Christopher J; Damha, Masad J; Parniak, Michael A

    2002-03-12

    Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially

  6. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

    PubMed Central

    Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

    2015-01-01

    MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

  7. Small antisense oligonucleotides against G-quadruplexes: specific mRNA translational switches

    PubMed Central

    Rouleau, Samuel G.; Beaudoin, Jean-Denis; Bisaillon, Martin; Perreault, Jean-Pierre

    2015-01-01

    G-quadruplexes (G4) are intricate RNA structures found throughout the transcriptome. Because they are associated with a variety of biological cellular mechanisms, these fascinating structural motifs are seen as potential therapeutic targets against many diseases. While screening of chemical compounds specific to G4 motifs has yielded interesting results, no single compound successfully discriminates between G4 motifs based on nucleotide sequences alone. This level of specificity is best attained using antisense oligonucleotides (ASO). Indeed, oligonucleotide-based strategies are already used to modulate DNA G4 folding in vitro. Here, we report that, in human cells, the use of short ASO to promote and inhibit RNA G4 folding affects the translation of specific mRNAs, including one from the 5′UTR of the H2AFY gene, a histone variant associated with cellular differentiation and cancer. These results suggest that the relatively high specificity of ASO-based strategies holds significant potential for applications aimed at modulating G4-motif folding. PMID:25510493

  8. Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

    PubMed Central

    Zhang, Ling; Yang, Xiang-dong; Zhang, Yuan-yu; Yang, Jing; Qi, Guang-xun; Guo, Dong-quan; Xing, Guo-jie; Yao, Yao; Xu, Wen-jing; Li, Hai-yun; Li, Qi-yun; Dong, Ying-shan

    2014-01-01

    The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts. PMID:25197629

  9. Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.

    PubMed

    Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke

    2016-03-01

    Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles.

  10. Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy

    PubMed Central

    Wojtkowiak-Szlachcic, Agnieszka; Taylor, Katarzyna; Stepniak-Konieczna, Ewa; Sznajder, Lukasz J.; Mykowska, Agnieszka; Sroka, Joanna; Thornton, Charles A.; Sobczak, Krzysztof

    2015-01-01

    Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3′-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUGexp) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUGexp/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUGexp foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUGexp-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1. PMID:25753670

  11. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models

    PubMed Central

    Osman, Erkan Y.; Miller, Madeline R.; Robbins, Kate L.; Lombardi, Abby M.; Atkinson, Arleigh K.; Brehm, Amanda J.; Lorson, Christian L.

    2014-01-01

    Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1MO-ASOs). A single intracerebroventricular injection in the relatively severe mouse model of SMA (SMNΔ7 mouse model) elicited a robust induction of SMN protein, and mean life span was extended from an average survival of 13 to 54 days following a single dose, consistent with large weight gains and a correction of the neuronal pathology. Additionally, E1MO-ASO treatment in an intermediate SMA mouse (SMNRT mouse model) significantly extended life span by ∼700% and weight gain was comparable with the unaffected animals. While a number of experimental therapeutics have targeted the ISS-N1 element of SMN2 pre-mRNA, the development of E1 ASOs provides a new molecular target for SMA therapeutics that dramatically extends survival in two important pre-clinical models of disease. PMID:24781211

  12. Regulating malonyl-CoA metabolism via synthetic antisense RNAs for enhanced biosynthesis of natural products.

    PubMed

    Yang, Yaping; Lin, Yuheng; Li, Lingyun; Linhardt, Robert J; Yan, Yajun

    2015-05-01

    Malonyl-CoA is the building block for fatty acid biosynthesis and also a precursor to various pharmaceutically and industrially valuable molecules, such as polyketides and biopolymers. However, intracellular malonyl-CoA is usually maintained at low levels, which poses great challenges to efficient microbial production of malonyl-CoA derived molecules. Inactivation of the malonyl-CoA consumption pathway to increase its intracellular availability is not applicable, since it is usually lethal to microorganisms. In this work, we employ synthetic antisense RNAs (asRNAs) to conditionally down-regulate fatty acid biosynthesis and achieve malonyl-CoA enrichment in Escherichia coli. The optimized asRNA constructs with a loop-stem structure exhibit high interference efficiency up to 80%, leading to a 4.5-fold increase in intracellular malonyl-CoA concentration when fabD gene expression is inhibited. Strikingly, this strategy allows the improved production of natural products 4-hydroxycoumarin, resveratrol, and naringenin by 2.53-, 1.70-, and 1.53-fold in E. coli, respectively. In addition, down-regulation of other fab genes including fabH, fabB, and fabF also leads to remarkable increases in 4-hydroxycoumarin production. This study demonstrates a novel strategy to enhance intracellular malonyl-CoA and indicates the effectiveness of asRNA as a powerful tool for use in metabolic engineering.

  13. In Vitro Inhibition of Hepatitis C Virus by Antisense Oligonucleotides in PBMC Compared to Hepatoma Cells

    PubMed Central

    Youssef, Samar Samir; Fahmy, Ahmed Mohamed; Omran, Moataza Hassan; Mohamed, Amr Saad; El Desouki, Mohamed Ali; El-Awady, Mostafa K.

    2014-01-01

    Aim. To assess the efficiency of phosphorothioate antisense oligodeoxynucleotide 1 (S-ODN1) on HCV translation inhibition in PBMC compared to hepatoma cells in vitro for the first time. Materials and Methods. The study included 34 treatment naive HCV patients. IRES domain III and IV sequence variations were tested in 45 clones from 9 HCV patients. PBMC of HCV positive patients were subjected to S-ODN in vitro. Concomitantly HepG2 cells infected by the same patient's serum were also treated with S-ODN1 for 24 and 48 hours. Cellular RNA was tested for HCV plus and minus strands by reverse transcription polymerase chain reaction (RT-PCR). Results. Sequence variations were seen in HCV IRES domain III only while domain IV was conserved among all the tested patient's clones. S-ODN1 successfully inhibited HCV translation in HepG2 cells, while in PBMC inhibition was partial. Conclusion. HCV IRES domain IV is more conserved than domain IIId in genotype 4 HCV patients. S-ODN against HCV IRES domain IV was not efficient to inhibit HCV translation in PBMC under the study conditions. Further studies testing other S-ODN targeting other HCV IRES domains in PBMC should be done. PMID:24991538

  14. Expression and regulation of the Msx1 natural antisense transcript during development.

    PubMed

    Coudert, Amélie E; Pibouin, Laurence; Vi-Fane, Brigitte; Thomas, Bethan L; Macdougall, Mary; Choudhury, Anuradha; Robert, Benoît; Sharpe, Paul T; Berdal, Ariane; Lezot, Frédéric

    2005-01-01

    Bidirectional transcription, leading to the expression of an antisense (AS) RNA partially complementary to the protein coding sense (S) RNA, is an emerging subject in mammals and has been associated with various processes such as RNA interference, imprinting and transcription inhibition. Homeobox genes do not escape this bidirectional transcription, raising the possibility that such AS transcription occurs during embryonic development and may be involved in the complexity of regulation of homeobox gene expression. According to the importance of the Msx1 homeobox gene function in craniofacial development, especially in tooth development, the expression and regulation of its recently identified AS transcripts were investigated in vivo in mouse from E9.5 embryo to newborn, and compared with the S transcript and the encoded protein expression pattern and regulation. The spatial and temporal expression patterns of S, AS transcripts and protein are consistent with a role of AS RNA in the regulation of Msx1 expression in timely controlled developmental sites. Epithelial-mesenchymal interactions were shown to control the spatial organization of S and also AS RNA expression during early patterning of incisors and molars in the odontogenic mesenchyme. To conclude, this study clearly identifies the Msx1 AS RNA involvement during tooth development and evidences a new degree of complexity in craniofacial developmental biology: the implication of endogenous AS RNAs.

  15. Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration.

    PubMed

    Babajko, Sylvie; Petit, Stéphane; Fernandes, Isabelle; Méary, Fleur; LeBihan, Johanne; Pibouin, Laurence; Berdal, Ariane

    2009-01-01

    Msx homeogenes play an important role in epithelial-mesenchymal interactions leading development. Msx1 is relevant for dental and craniofacial morphogenesis, as suggested by phenotypes of Msx1 mutations in human and Msx1 KO mice. During adulthood, Msx1 is still expressed in the skeleton where its role is largely unknown. Our group showed that the Msx1 gene is submitted to bidirectional transcription generating a long noncoding antisense (AS) RNA. During tooth development, Msx1 sense (S) and AS RNAs showed specific patterns of expression. Thus, the aim of the present study was to analyze the relation between Msx1 S and AS RNAs. In vivo mapping in adult mice showed that both Msx1 RNAs were detected in tested tissues such as bone. In vitro, Msx1 AS RNA decreased endogenous Msx1 S expression and modified Msx1 protein cell distribution. Regulations of Dlx5 and Bmp4 expression involving Msx1 S and AS RNAs showed that Msx1 AS RNA could modulate Msx1 function. The study of Msx1 S and AS RNA status is interesting in the case of tooth agenesis and bone loss to see if a disturbance of this balance could be associated with a disturbance of bone homeostasis. In that sense, our current results suggest a clear involvement of Msx1 in alveolar bone.

  16. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  17. Thiolated polycarbophil as an adjuvant for permeation enhancement in nasal delivery of antisense oligonucleotides.

    PubMed

    Vetter, A; Martien, R; Bernkop-Schnürch, A

    2010-03-01

    The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO-ODN) on the nasal mucosa. Polycarbophil-cysteine (PCP-Cys) was synthesized by the covalent attachment of L-cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO-ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP-cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing-type diffusion chambers. PCP-Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO-ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO-ODNs from the nasal mucosa was 1.7-fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO-ODNs.

  18. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    PubMed

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  19. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3

    PubMed Central

    Toonen, Lodewijk J. A.; Schmidt, Iris; Luijsterburg, Martijn S.; van Attikum, Haico; van Roon-Mom, Willeke M. C.

    2016-01-01

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3. PMID:27731380

  20. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

    PubMed Central

    Gramlich, Michael; Pane, Luna Simona; Zhou, Qifeng; Chen, Zhifen; Murgia, Marta; Schötterl, Sonja; Goedel, Alexander; Metzger, Katja; Brade, Thomas; Parrotta, Elvira; Schaller, Martin; Gerull, Brenda; Thierfelder, Ludwig; Aartsma-Rus, Annemieke; Labeit, Siegfried; Atherton, John J; McGaughran, Julie; Harvey, Richard P; Sinnecker, Daniel; Mann, Matthias; Laugwitz, Karl-Ludwig; Gawaz, Meinrad Paul; Moretti, Alessandra

    2015-01-01

    Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM. PMID:25759365

  1. Antibacterial activity and inhibition of protein synthesis in Escherichia coli by antisense DNA analogs.

    PubMed

    Rahman, M A; Summerton, J; Foster, E; Cunningham, K; Stirchak, E; Weller, D; Schaup, H W

    1991-01-01

    Protein synthesis, which takes place within ribosomes, is essential for the survival of any living organism. Ribosomes are composed of both proteins and RNA. Specific interaction between the 3' end CCUCC sequence of prokaryotic 16S rRNA and a partially complementary sequence preceding the initiating codon of mRNA is believed to be a prerequisite for initiation of protein synthesis. Here we report the use of short (three to six nucleotides) synthetic DNA analogs complementary to this sequence to block protein synthesis in vitro and in vivo in Escherichia coli. In the DNA analogs the normal phosphodiester bond in the antisense DNA was replaced by methylcarbamate internucleoside linkages to enhance transport across plasma membranes. Of the analogs tested, those with the sequence AGG and GGA inhibit protein synthesis and colony formation by E. coli strains lacking an outer cell wall. Polyethylene glycol 1000 (PEG 1000) was attached to the 5' end of some of the test methylcarbamate DNAs to enhance solubility. Analogs of AGG and GGAG with PEG 1000 attached inhibited colony formation in normal E. coli. These analogs may be useful food additives to control bacterial spoilage and biomedically as antibiotics. PMID:1821653

  2. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-07-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

  3. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

    NASA Technical Reports Server (NTRS)

    Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

    2003-01-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

  4. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    PubMed

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  5. Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.

    PubMed

    Zhang, Ling; Yang, Xiang-Dong; Zhang, Yuan-Yu; Yang, Jing; Qi, Guang-Xun; Guo, Dong-Quan; Xing, Guo-Jie; Yao, Yao; Xu, Wen-Jing; Li, Hai-Yun; Li, Qi-Yun; Dong, Ying-Shan

    2014-01-01

    The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

  6. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    PubMed

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  7. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    PubMed Central

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  8. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

    PubMed Central

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

    2016-01-01

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

  9. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana.

    PubMed

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-06-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications. PMID:25922535

  10. Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages

    PubMed Central

    Wan, W. Brad; Migawa, Michael T.; Vasquez, Guillermo; Murray, Heather M.; Nichols, Josh G.; Gaus, Hans; Berdeja, Andres; Lee, Sam; Hart, Christopher E.; Lima, Walt F.; Swayze, Eric E.; Seth, Punit P.

    2014-01-01

    Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability. PMID:25398895

  11. Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes.

    PubMed Central

    Crooke, S T; Lemonidis, K M; Neilson, L; Griffey, R; Lesnik, E A; Monia, B P

    1995-01-01

    1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8526876

  12. Effect of Antisense Oligodeoxynucleotides Glucose Transporter-1 on Enhancement of Radiosensitivity of Laryngeal Carcinoma

    PubMed Central

    Yan, Sen-Xiang; Luo, Xing-Mei; Zhou, Shui-Hong; Bao, Yang-Yang; Fan, Jun; Lu, Zhong-Jie; Liao, Xin-Biao; Huang, Ya-Ping; Wu, Ting-Ting; Wang, Qin-Ying

    2013-01-01

    Purpose: Laryngeal carcinomas always resist to radiotherapy. Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is considered to be a possible intrinsic marker of hypoxia in malignant tumors. We speculated that the inhibition of GLUT-1 expression might improve the radiosensitivity of laryngeal carcinoma. Methods: We assessed the effect of GLUT-1 expression on radioresistance of laryngeal carcinoma and the effect of GLUT-1 expressions by antisense oligodeoxynucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma in vitro and in vivo. Results: After transfection of GLUT-1 AS-ODNs: MTS assay showed the survival rates of radiation groups were reduced with the prolongation of culture time (p<0.05); Cell survival rates were significantly reduced along with the increasing of radiation dose (p<0.05). There was significant difference in the expression of GLUT-1mRNA and protein in the same X-ray dose between before and after X-ray radiation (p<0.05). In vivo, the expressions of GLUT-1 mRNA and protein after 8Gy radiation plus transfection of GLUT-1 AS-ODNs were significant decreased compared to 8Gy radiation alone (p<0.001). Conclusion: Radioresistance of laryngeal carcinoma may be associated with increased expression of GLUT-1 mRNA and protein. GLUT-1 AS-ODNs may enhance the radiosensitivity of laryngeal carcinoma mainly by inhibiting the expression of GLUT-1. PMID:23983599

  13. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

    PubMed

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  14. Biophysical and biological characterization of hairpin and molecular beacon RNase H active antisense oligonucleotides.

    PubMed

    Østergaard, Michael E; Thomas, George; Koller, Erich; Southwell, Amber L; Hayden, Michael R; Seth, Punit P

    2015-05-15

    Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA in vitro, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice.

  15. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides

    PubMed Central

    Kasuya, Takeshi; Hori, Shin-ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer’s gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  16. Stress-induced glucocorticoids suppress the antisense molecular regulation of FGF-2 expression.

    PubMed

    Frank, Matthew G; Der-Avakian, Andre; Bland, Sondra T; Watkins, Linda R; Maier, Steven F

    2007-05-01

    Psychological stress can upregulate basic fibroblast growth factor (FGF-2) expression. Because glucocorticoids can also upregulate FGF-2 expression, the present studies investigated whether stress-induced glucocorticoids mediate the effects of stress on FGF-2. FGF-2 is regulated by an FGF-2 antisense (AS) molecular mechanism and so the present experiments also, for the first time, assessed the effects of stress on FGF-2-AS mRNA, as well as the mediating role of glucocorticoids. The effects of either escapable shock (ES) or yoked-inescapable tail shock (IS) on FGF-2 and FGF-2-AS were determined. To test whether glucocorticoids mediate the effect of stress on FGF-2 and FGF-2-AS, animals were pretreated with temporary corticosterone (CORT) synthesis inhibitors and exposed to IS. To test whether glucocorticoids are sufficient to modulate FGF-2 and FGF-2-AS mRNA, animals were injected with CORT and mRNA measured. ES and IS similarly downregulated FGF-2-AS mRNA at 0 h post-stress and upregulated FGF-2 mRNA 2 h post-stress. Inhibition of CORT synthesis abrogated the effect of IS on both FGF-2-AS and FGF-2 mRNA. Exogenous CORT mimicked the effects of ES and IS on FGF-2, but not FGF-2-AS mRNA. The present study demonstrates that glucocorticoids mediate the effects of stress on FGF-2 and FGF-2-AS.

  17. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana.

    PubMed

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-06-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications.

  18. Proteome changes in tomato lines transformed with phytoene synthase-1 in the sense and antisense orientations.

    PubMed

    Robertson, Francesca P; Koistinen, P Kaisa; Gerrish, Christopher; Halket, John M; Patel, Raj K P; Fraser, Paul D; Bramley, Peter M

    2012-10-01

    The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.

  19. Autolysis of cell walls from polygalacturonase-antisense tomato fruit in simulated apoplastic solutions.

    PubMed

    Almeida, Domingos P F; Huber, Donald J

    2011-06-01

    Autolysis of cell walls from polygalacturonase (PG)-antisense tomato fruit was studied in a conventional buffer designed to maximize the catalytic activity of PG (30 mM sodium acetate, 150 mM NaCl, pH 4.5), and in solutions mimicking the pH and mineral composition of the fruit apoplast at the mature-green and ripe stages. Autolytic release of uronic acids was very limited under simulated apoplastic conditions compared with the conventional buffer, but minimal differences in the release of reducing groups were observed among the incubation conditions. Autolytic release of uronic acids from active walls was lower than solubilization from enzymically inactive walls. Uronic acids that remained ionically bound to the cell walls during autolysis were subsequently extracted and analyzed by size exclusion chromatography. The elution profiles of ionically bound uronic acids from cell walls incubated under optimal conditions were similar for all ripening stages. In solutions mimicking the pH and mineral composition of the apoplast of mature-green and ripe fruit, uronic acids extracted from pink and ripe fruit cell walls showed a decrease in average molecular mass compared with polymers from mature-green cell walls. The results suggest that the composition of the incubation solution exert strong influence on PG-independent cell wall autolysis and that enzymically active walls restrain PG-independent pectin solubilization.

  20. Predictive Dose-Based Estimation of Systemic Exposure Multiples in Mouse and Monkey Relative to Human for Antisense Oligonucleotides With 2′-O-(2-Methoxyethyl) Modifications

    PubMed Central

    Yu, Rosie Z; Grundy, John S; Henry, Scott P; Kim, Tae-Won; Norris, Daniel A; Burkey, Jennifer; Wang, Yanfeng; Vick, Andrew; Geary, Richard S

    2015-01-01

    Evaluation of species differences and systemic exposure multiples (or ratios) in toxicological animal species versus human is an ongoing exercise during the course of drug development. The systemic exposure ratios are best estimated by directly comparing area under the plasma concentration-time curves (AUCs), and sometimes by comparing the dose administered, with the dose being adjusted either by body surface area (BSA) or body weight (BW). In this study, the association between AUC ratio and the administered dose ratio from animals to human were studied using a retrospective data-driven approach. The dataset included nine antisense oligonucleotides (ASOs) with 2′-O-(2-methoxyethyl) modifications, evaluated in two animal species (mouse and monkey) following single and repeated parenteral administrations. We found that plasma AUCs were similar between ASOs within the same species, and are predictable to human exposure using a single animal species, either mouse or monkey. Between monkey and human, the plasma exposure ratio can be predicted directly based on BW-adjusted dose ratios, whereas between mouse and human, the exposure ratio would be nearly fivefold lower in mouse compared to human based on BW-adjusted dose values. Thus, multiplying a factor of 5 for the mouse BW-adjusted dose would likely provide a reasonable AUC exposure estimate in human at steady-state. PMID:25602582

  1. Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.

    PubMed

    Thumma, S C; Jacobson, B A; Patel, M R; Konicek, B W; Franklin, M J; Jay-Dixon, J; Sadiq, A; De, A; Graff, J R; Kratzke, R A

    2015-08-01

    Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

  2. Predictive dose-based estimation of systemic exposure multiples in mouse and monkey relative to human for antisense oligonucleotides with 2'-o-(2-methoxyethyl) modifications.

    PubMed

    Yu, Rosie Z; Grundy, John S; Henry, Scott P; Kim, Tae-Won; Norris, Daniel A; Burkey, Jennifer; Wang, Yanfeng; Vick, Andrew; Geary, Richard S

    2015-01-20

    Evaluation of species differences and systemic exposure multiples (or ratios) in toxicological animal species versus human is an ongoing exercise during the course of drug development. The systemic exposure ratios are best estimated by directly comparing area under the plasma concentration-time curves (AUCs), and sometimes by comparing the dose administered, with the dose being adjusted either by body surface area (BSA) or body weight (BW). In this study, the association between AUC ratio and the administered dose ratio from animals to human were studied using a retrospective data-driven approach. The dataset included nine antisense oligonucleotides (ASOs) with 2'-O-(2-methoxyethyl) modifications, evaluated in two animal species (mouse and monkey) following single and repeated parenteral administrations. We found that plasma AUCs were similar between ASOs within the same species, and are predictable to human exposure using a single animal species, either mouse or monkey. Between monkey and human, the plasma exposure ratio can be predicted directly based on BW-adjusted dose ratios, whereas between mouse and human, the exposure ratio would be nearly fivefold lower in mouse compared to human based on BW-adjusted dose values. Thus, multiplying a factor of 5 for the mouse BW-adjusted dose would likely provide a reasonable AUC exposure estimate in human at steady-state.

  3. miR-Synth: a computational resource for the design of multi-site multi-target synthetic miRNAs

    PubMed Central

    Laganà, Alessandro; Acunzo, Mario; Romano, Giulia; Pulvirenti, Alfredo; Veneziano, Dario; Cascione, Luciano; Giugno, Rosalba; Gasparini, Pierluigi; Shasha, Dennis; Ferro, Alfredo; Croce, Carlo Maria

    2014-01-01

    RNAi is a powerful tool for the regulation of gene expression. It is widely and successfully employed in functional studies and is now emerging as a promising therapeutic approach. Several RNAi-based clinical trials suggest encouraging results in the treatment of a variety of diseases, including cancer. Here we present miR-Synth, a computational resource for the design of synthetic microRNAs able to target multiple genes in multiple sites. The proposed strategy constitutes a valid alternative to the use of siRNA, allowing the employment of a fewer number of molecules for the inhibition of multiple targets. This may represent a great advantage in designing therapies for diseases caused by crucial cellular pathways altered by multiple dysregulated genes. The system has been successfully validated on two of the most prominent genes associated to lung cancer, c-MET and Epidermal Growth Factor Receptor (EGFR). (See http://microrna.osumc.edu/mir-synth). PMID:24627222

  4. Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.

    PubMed Central

    Wagner, J A; McDonald, T V; Nghiem, P T; Lowe, A W; Schulman, H; Gruenert, D C; Stryer, L; Gardner, P

    1992-01-01

    Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway. Images PMID:1379720

  5. Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

    PubMed

    Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

    2016-09-01

    Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

  6. Antisense against Amyloid-β Protein Precursor Reverses Memory Deficits and Alters Gene Expression in Neurotropic and Insulin-Signaling Pathways in SAMP8 Mice.

    PubMed

    Armbrecht, Harvey J; Siddiqui, Akbar M; Green, Michael; Farr, Susan A; Kumar, Vijaya B; Banks, William A; Patrick, Ping; Shah, Gul N; Morley, John E

    2015-01-01

    The senescence-accelerated mouse (SAMP8) strain exhibits an age-related decrease in memory accompanied by an increase in hippocampal amyloid-β protein precursor (AβPP) and amyloid-β peptide (Aβ). We have shown that administration of an antisense oligonucleotide against the Aβ region of AβPP (AβPP antisense) reverses the memory deficits. The purpose of this study was to determine the effect of peripheral (IV) administration of AβPP antisense on hippocampal gene expression. The AβPP antisense reversed the memory deficits and altered expression of 944 hippocampal genes. Pathway analysis showed significant gene expression changes in nine pathways. These include the MAPK signaling pathway (p = 0.0078) and the phosphatidylinositol signaling pathway (p = 0.043), which we have previously shown to be altered in SAMP8 mice. The changes in these pathways contributed to significant changes in the neurotropin (p = 0.0083) and insulin signaling (p = 0.015) pathways, which are known to be important in learning and memory. Changes in these pathways were accompanied by phosphorylation changes in the downstream target proteins p70S6K, GSK3β, ERK, and CREB. These changes in hippocampal gene expression and protein phosphorylation may suggest specific new targets for antisense therapy aimed at improving memory.

  7. Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

    PubMed Central

    Sullenger, B A; Lee, T C; Smith, C A; Ungers, G E; Gilboa, E

    1990-01-01

    NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies. Images PMID:2247070

  8. Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys

    PubMed Central

    Shemesh, Colby S; Yu, Rosie Z; Gaus, Hans J; Greenlee, Sarah; Post, Noah; Schmidt, Karsten; Migawa, Michael T; Seth, Punit P; Zanardi, Thomas A; Prakash, Thazha P; Swayze, Eric E; Henry, Scott P; Wang, Yanfeng

    2016-01-01

    Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of 3H-radiolabeled (3H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5′-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs. PMID:27164023

  9. The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

    PubMed Central

    Heinrich, J; Riedel, H D; Rückert, B; Lurz, R; Schuster, H

    1995-01-01

    The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon. Images PMID:7784198

  10. Sense-antisense gene-pairs in breast cancer and associated pathological pathways

    PubMed Central

    Grinchuk, Oleg V.; Motakis, Efthymios; Yenamandra, Surya Pavan; Ow, Ghim Siong; Jenjaroenpun, Piroon; Tang, Zhiqun; Yarmishyn, Aliaksandr A.; Ivshina, Anna V.; Kuznetsov, Vladimir A.

    2015-01-01

    More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers. PMID:26517092

  11. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  12. Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene1

    PubMed Central

    Jiménez-Bermúdez, Silvia; Redondo-Nevado, José; Muñoz-Blanco, Juan; Caballero, José L.; López-Aranda, José M.; Valpuesta, Victoriano; Pliego-Alfaro, Fernando; Quesada, Miguel A.; Mercado, José A.

    2002-01-01

    Strawberry (Fragaria × ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry. PMID:11842178

  13. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    SciTech Connect

    Feng Xian Lu; Aiyar, N.; Chaiken, I. )

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  14. Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease.

    PubMed

    Ripple, Michael J; You, Dahui; Honnegowda, Srinivasa; Giaimo, Joseph D; Sewell, Andrew B; Becnel, David M; Cormier, Stephania A

    2010-10-15

    Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Severe RSV infections in infants cause bronchiolitis, wheeze, and/or cough and significantly increase the risk for developing asthma. RSV pathogenesis is thought to be due to a Th2-type immune response initiated in response to RSV infection, specifically in the infant. Using a neonatal mouse system as an appropriate model for human infants, we sought to determine whether local inhibition of IL-4Rα expression during primary RSV infection in the neonate would prevent Th2-skewed responses to secondary RSV infection and improve long-term pulmonary function. To reduce IL-4Rα expression, antisense oligonucleotides (ASOs) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at 1 wk of age and were reinfected at 6 wk of age. Administration of IL-4Rα ASOs during primary RSV infection in neonatal mice abolished the pulmonary dysfunction normally observed following reinfection in the adult. This ablation of pulmonary dysfunction correlated with a persistent rebalancing of the Th cell compartment with decreased Th2 responses (i.e., reduced goblet cell hyperplasia, Th2 cells, and cytokine secretion) and increased Th1 responses (i.e., elevated Th1 cell numbers and type I Abs and cytokines). Our data support our hypothesis that a reduction in the Th2 immune response during primary infection in neonates prevents Th2-mediated pulmonary pathology initially and upon reinfection and further suggest that vaccine strategies incorporating IL-4Rα ASOs may be of significant benefit to infants.

  15. Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models

    SciTech Connect

    Roedel, Franz; Capalbo, Gianni; Weiss, Christian; Roedel, Claus

    2008-05-01

    Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

  16. Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

    PubMed

    Quan, S; Yang, L; Abraham, N G; Kappas, A

    2001-10-01

    Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

  17. Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys.

    PubMed

    Shemesh, Colby S; Yu, Rosie Z; Gaus, Hans J; Greenlee, Sarah; Post, Noah; Schmidt, Karsten; Migawa, Michael T; Seth, Punit P; Zanardi, Thomas A; Prakash, Thazha P; Swayze, Eric E; Henry, Scott P; Wang, Yanfeng

    2016-01-01

    Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs. PMID:27164023

  18. A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse

    PubMed Central

    Porensky, Paul N.; Mitrpant, Chalermchai; McGovern, Vicki L.; Bevan, Adam K.; Foust, Kevin D.; Kaspar, Brain K.; Wilton, Stephen D.; Burghes, Arthur H.M.

    2012-01-01

    Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(−10,−29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn−/−, SMN2+/+, SMN▵7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients. PMID:22186025

  19. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.

    PubMed

    Seth, Punit P; Jazayeri, Ali; Yu, Jeff; Allerson, Charles R; Bhat, Balkrishen; Swayze, Eric E

    2012-01-01

    We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012. PMID:23344239

  20. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

    PubMed Central

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies. PMID:27124114

  1. Design of multi-target compounds as AChE, BACE1, and amyloid-β(1-42) oligomerization inhibitors: in silico and in vitro studies.

    PubMed

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Martínez-Ramos, Federico; Padilla-Martínez, Itzia Irene; Benítez-Cardoza, Claudia G; Mera-Jiménez, Elvia; Rosales-Hernández, Martha Cecilia

    2014-01-01

    Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models.

  2. Design of multi-target compounds as AChE, BACE1, and amyloid-β(1-42) oligomerization inhibitors: in silico and in vitro studies.

    PubMed

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Martínez-Ramos, Federico; Padilla-Martínez, Itzia Irene; Benítez-Cardoza, Claudia G; Mera-Jiménez, Elvia; Rosales-Hernández, Martha Cecilia

    2014-01-01

    Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models. PMID:24762947

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