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Sample records for multi-target antisense approach

  1. Antisense approaches in prostate cancer.

    PubMed

    Chi, Kim N; Gleave, Martin E

    2004-06-01

    Patients with hormone refractory prostate cancer have limited treatment options and new therapies are urgently needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets that are involved in apoptosis, growth factors, cell signalling and the androgen receptor (AR). Antisense oligonucleotides are short sequences of synthetic modified DNA that are designed to be complimentary to a selected gene's mRNA and thereby specifically inhibit expression of that gene. The antisense approach continues to hold promise as a therapeutic modality to target genes involved in cancer progression, especially those in which the gene products are not amenable to small molecule inhibition or antibodies. The current status and future direction of a number of antisense oligonucleotides targeting several genes, including BCL-2, BCL-XL, clusterin, the inhibitors of apoptosis (IAP) family, MDM2, protein kinase C-alpha, c-raf, insulin-like growth factor binding proteins and the AR, that have potential clinical use in prostate cancer are reviewed. PMID:15174974

  2. 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

  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. A multi target approach to control chemical reactions in their inhomogeneous solvent environment

    NASA Astrophysics Data System (ADS)

    Keefer, Daniel; Thallmair, Sebastian; Zauleck, Julius P. P.; de Vivie-Riedle, Regina

    2015-12-01

    Shaped laser pulses offer a powerful tool to manipulate molecular quantum systems. Their application to chemical reactions in solution is a promising concept to redesign chemical synthesis. Along this road, theoretical developments to include the solvent surrounding are necessary. An appropriate theoretical treatment is helpful to understand the underlying mechanisms. In our approach we simulate the solvent by randomly selected snapshots from molecular dynamics trajectories. We use multi target optimal control theory to optimize pulses for the various arrangements of explicit solvent molecules simultaneously. This constitutes a major challenge for the control algorithm, as the solvent configurations introduce a large inhomogeneity to the potential surfaces. We investigate how the algorithm handles the new challenges and how well the controllability of the system is preserved with increasing complexity. Additionally, we introduce a way to statistically estimate the efficiency of the optimized laser pulses in the complete thermodynamical ensemble.

  5. Combined analgesics in (headache) pain therapy: shotgun approach or precise multi-target therapeutics?

    PubMed Central

    2011-01-01

    Background Pain in general and headache in particular are characterized by a change in activity in brain areas involved in pain processing. The therapeutic challenge is to identify drugs with molecular targets that restore the healthy state, resulting in meaningful pain relief or even freedom from pain. Different aspects of pain perception, i.e. sensory and affective components, also explain why there is not just one single target structure for therapeutic approaches to pain. A network of brain areas ("pain matrix") are involved in pain perception and pain control. This diversification of the pain system explains why a wide range of molecularly different substances can be used in the treatment of different pain states and why in recent years more and more studies have described a superior efficacy of a precise multi-target combination therapy compared to therapy with monotherapeutics. Discussion In this article, we discuss the available literature on the effects of several fixed-dose combinations in the treatment of headaches and discuss the evidence in support of the role of combination therapy in the pharmacotherapy of pain, particularly of headaches. The scientific rationale behind multi-target combinations is the therapeutic benefit that could not be achieved by the individual constituents and that the single substances of the combinations act together additively or even multiplicatively and cooperate to achieve a completeness of the desired therapeutic effect. As an example the fixesd-dose combination of acetylsalicylic acid (ASA), paracetamol (acetaminophen) and caffeine is reviewed in detail. The major advantage of using such a fixed combination is that the active ingredients act on different but distinct molecular targets and thus are able to act on more signalling cascades involved in pain than most single analgesics without adding more side effects to the therapy. Summary Multitarget therapeutics like combined analgesics broaden the array of therapeutic

  6. Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach.

    PubMed

    Chandolia, Amita; Rathor, Nisha; Sharma, Monika; Saini, Neeraj Kumar; Sinha, Rajesh; Malhotra, Pawan; Brahmachari, Vani; Bose, Mridula

    2014-01-01

    Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy. PMID:24556072

  7. Antisense approach to inflammatory bowel disease: prospects and challenges.

    PubMed

    Marafini, Irene; Di Fusco, Davide; Calabrese, Emma; Sedda, Silvia; Pallone, Francesco; Monteleone, Giovanni

    2015-05-01

    Despite the great success of anti-tumour necrosis factor-based therapies, the treatment of Crohn's disease (CD) and ulcerative colitis (UC) still remains a challenge for clinicians, as these drugs are not effective in all patients, their efficacy may wane with time, and their use can increase the risk of adverse events and be associated with the development of new immune-mediated diseases. Therefore, new therapeutic targets are currently being investigated both in pre-clinical studies and in clinical trials. Among the technologies used to build new therapeutic compounds, the antisense oligonucleotide (ASO) approach is slowly gaining space in the field of inflammatory bowel diseases (IBDs), and three ASOs have been investigated in clinical trials. Systemic administration of alicaforsen targeting intercellular adhesion molecule-1, a protein involved in the recruitment of leukocytes to inflamed intestine, was not effective in CD, even though the same compound was of benefit when given as an enema to UC patients. DIMS0150, targeting nuclear factor (NF) κB-p65, a transcription factor that promotes pro-inflammatory responses, was very promising in pre-clinical studies and is currently being tested in clinical trials. Oral mongersen, targeting Smad7, an intracellular protein that inhibits transforming growth factor (TGF)-β1 activity, was safe and well tolerated by CD patients, and the results of a phase II clinical trial showed the efficacy of the drug in inducing clinical remission in patients with active disease. In this leading article, we review the rationale and the clinical data available regarding these three agents, and we discuss the challenge of using ASOs in IBD. PMID:25911184

  8. Epigenetic-based therapy: From single- to multi-target approaches.

    PubMed

    Benedetti, Rosaria; Conte, Mariarosaria; Iside, Concetta; Altucci, Lucia

    2015-12-01

    The treatment of cancer has traditionally been based on the identification of a single molecule and/or enzymatic function (target) responsible for a particular phenotype, and therefore on the ability to stimulate, attenuate or inhibit its activity through the use of selective compounds. However, cancer is no longer considered a disease caused by a single factor, but is now recognized as a multi-factorial disorder. Genetic, epigenetic and metabolic factors all contribute to neoplasia, causing significant changes in molecular networks that govern cell growth, development, death and specialization. Consequently, many antitumor therapies are no longer directed against a single target but the biological system as a whole, in which functions determining the onset and maintenance of a physio-pathological state are modulated. The field of epi-drug discovery is currently in a transitional phase where the search for putative anticancer drugs is shifting from single-target-oriented molecules to network-active compounds and to epi-drugs used in combination with other epi-agents and with traditional chemotherapeutics. This review illustrates the pros and cons of each therapeutic option, providing examples in support of single-target and multi (network)-target epi-drug approaches. PMID:26494003

  9. Ilizarov Treatment of Congenital Pseudarthrosis of the Tibia: A Multi-Targeted Approach Using the Ilizarov Technique

    PubMed Central

    Cho, Tae-Joon; Moon, Hyuk Ju

    2011-01-01

    Congenital pseudarthrosis of the tibia (CPT) is one of the most challenging problems in pediatric orthopaedics. The treatment goals are osteosynthesis, stabilization of the ankle mortise by fibular stabilization, and lower limb-length equalization. Each of these goals is difficult to accomplish but regardless of the surgical options, the basic biological considerations are the same: pseudarthrosis resection, biological bone bridging of the defect by stable fixation, and the correction of any angular deformity. The Ilizarov method is certainly valuable for the treatment of CPT because it can address not only pseudarthrosis but also all complex deformities associated with this condition. Leg-length discrepancy can be managed by proximal tibial lengthening using distraction osteogenesis combined with or without contralateral epiphysiodesis. However, treatment of CPT is fraught with complications due to the complex nature of the disease, and failure is common. Residual challenges, such as refracture, growth disturbance, and poor foot and ankle function with stiffness, are frequent and perplexing. Refracture is the most common and serious complication after primary healing and might result in the re-establishment of pseudarthrosis. Therefore, an effective, safe and practical treatment method that minimizes the residual challenges after healing and accomplishes the multiple goals of treatment is needed. This review describes a multi-targeted approach for tackling these challenges, which utilizes the Ilizarov technique in atrophic-type CPT. PMID:21369472

  10. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. PMID:22064367

  11. 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

  12. Antisense Therapy in Neurology

    PubMed Central

    Lee, Joshua J.A.; Yokota, Toshifumi

    2013-01-01

    Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy (including limb-girdle muscular dystrophy 2B; LGMD2B, Miyoshi myopathy; MM, and distal myopathy with anterior tibial onset; DMAT), and myotonic dystrophy (DM) are all reported to be promising targets for antisense therapy. This paper focuses on the current progress of antisense therapies in neurology. PMID:25562650

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. Morpholinos: Antisense and Sensibility.

    PubMed

    Blum, Martin; De Robertis, Edward M; Wallingford, John B; Niehrs, Christof

    2015-10-26

    For over 15 years, antisense morpholino oligonucleotides (MOs) have allowed developmental biologists to make key discoveries regarding developmental mechanisms in numerous model organisms. Recently, serious concerns have been raised as to the specificity of MO effects, and it has been recommended to discontinue their usage, despite the long experience of the scientific community with the MO tool in thousands of studies. Reviewing the many advantages afforded by MOs, we conclude that adequately controlled MOs should continue to be accepted as generic loss-of-function approach, as otherwise progress in developmental biology will greatly suffer. PMID:26506304

  1. 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.

  2. 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

  3. 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.

  4. A stochastic grid filter for multi-target tracking

    NASA Astrophysics Data System (ADS)

    Kim, Surrey; Kouritzin, Michael A.; Long, Hongwei; McCrosky, Jesse D.; Zhao, Xingqiu

    2004-08-01

    In this paper, we discuss multi-target tracking for a submarine model based on incomplete observations. The submarine model is a weakly interacting stochastic dynamic system with several submarines in the underlying region. Observations are obtained at discrete times from a number of sonobuoys equipped with hydrophones and consist of a nonlinear function of the current locations of submarines corrupted by additive noise. We use filtering methods to find the best estimation for the locations of the submarines. Our signal is a measure-valued process, resulting in filtering equations that can not be readily implemented. We develop Markov chain approximation approach to solve the filtering equation for our model. Our Markov chains are constructed by dividing the multi-target state space into cells, evolving particles in these cells, and employing a random time change approach. These approximations converge to the unnormalized conditional distribution of the signal process based on the back observations. Finally we present some simulation results by using the refining stochastic grid (REST) filter (developed from our Markov chain approximation method).

  5. 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.

  6. An intelligent multi-target tracking system

    NASA Astrophysics Data System (ADS)

    Heyerdahl, E.

    1987-07-01

    An implementation of a general tracking system, integrating the target acquisition and tracking subsystems, was developed. It is based on image analysis and extensive use of models. The system permits improvements compared to in-service trackers in the sense that it enables multi-target tracking, automatic acquisition also during tracking and tracking through obscurations. The system is an implementation of a general tracking system. This system produces alternative estimates of a target and projects the corresponding objects into the image plane. To do this estimates of the projecting function are used. The different projections are synthesized through a thresholding process. The implemented system uses parallel Kalman filters to produce the object estimates and estimates the sensor position through a model of sensor dynamics and measurements of sensor angle velocity. Results, produced by the implemented system from IR imagery of a moving target in field are presented.

  7. 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)

  8. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

    The continued progress in our understanding of the biology of neoplasia and in the identification, cloning, and sequencing of genes critical to tumor cell function permits the exploitation of this information to develop specific agents that may directly modulate the function of these genes or their protein products. Antisense oligonucleotides are being investigated as a potential therapeutic modality that takes direct advantage of molecular sequencing. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA transcript through Watson-Crick base pairing. The formation of this oligonucleotide: RNA heteroduplex results in mRNA inactivation and consequent inhibition of synthesis of the protein product. A fundamental attraction of the antisense approach is that this method potentially may be applied to any gene product, in theory, for the treatment of malignant and non-malignant diseases. However, this simple and attractive model has proven to be much more complex in practice. A number of important challenges in the preclinical development of antisense oligonucleotides have been identified, including stability, sequence length, cellular uptake, target sequence selection, appropriate negative controls, oligonucleotide: protein interactions, and cost of manufacture. Although the biological activity of an oligonucleotide against its molecular target is theoretically sequence-dependent, the animal pharmacokinetics and toxicology of phosphorothioate analogues directed against vastly disparate gene products appear relatively non-sequence-specific. In oncology, a number of clinical trials have been initiated with antisense oligonucleotides directed against molecular targets including: p53; bcl-2; raf kinase; protein kinase C-alpha; c-myb. The experience gained from these early clinical trials will be applicable to the next generation of antisense agents in development. These may include molecules with novel backbones or other structural

  9. 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.

  10. Multi-target compressive laser ranging

    NASA Astrophysics Data System (ADS)

    Pandit, Pushkar P.; Dahl, Jason R.; Barber, Zeb W.; Babbitt, W. Randall

    2014-05-01

    Compressive laser ranging (CLR) is a method that exploits the sparsity available in the range domain using compressive sensing methods to directly obtain range domain information. Conventional ranging methods are marred by requirements of high bandwidth analog detection which includes severe SNR fall off with bandwidth in analog-to-digital conversion (ADC). Compressive laser ranging solves this problem by obtaining sub-centimeter resolution while using low bandwidth detection. High rate digital pulse pattern generators and off the shelf photonic devices are used to modulate the transmitted and received light from a superluminescent diode. CLR detection is demonstrated using low bandwidth, high dynamic range detectors along with photon counting techniques. The use of an incoherent source eliminates speckle issues and enables simplified CLR methods to get multi-target range profiles with 1-3cm resolution. Using compressive sensing methods CLR allows direct range measurements in the sub-Nyquist regime while reducing system resources, in particular the need for high bandwidth ADC.

  11. 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.

  12. Role of sialosyl Lewis(a) in adhesion of colon cancer cells--the antisense RNA approach.

    PubMed

    Kłopocki, A G; Laskowska, A; Antoniewicz-Papis, J; Duk, M; Lisowska, E; Ugorski, M

    1998-04-01

    To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Le(a) antigen we created a specific loss-of-function phenotype. A stable subclone (CX-1.1) with high expression of sialosyl Le(a) structure, obtained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDNA for alpha1,3/4-fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Le(a) carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjugates carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX-1.1 cells were analysed for the expression of this structure. Anti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an intense band representing sialosyl Le(a)-ganglioside on a thin-layer chromatogram. Using O-sialoglycoprotease from Pasteurella haemolytica and an alkaline beta-elimination procedure, it was shown that protein-linked sialosyl Le(a) structures are carried mostly by mucin-type glycoproteins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese hamster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was confirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O

  13. 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

  14. 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

  15. 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

  16. 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

  17. The anti-dementia drug candidate, (-)-clausenamide, improves memory impairment through its multi-target effect.

    PubMed

    Chu, Shifeng; Liu, Shaolin; Duan, Wenzhen; Cheng, Yong; Jiang, Xueying; Zhu, Chuanjiang; Tang, Kang; Wang, Runsheng; Xu, Lin; Wang, Xiaoying; Yu, Xiaoming; Wu, Kemei; Wang, Yan; Wang, Muzou; Huang, Huiyong; Zhang, Juntian

    2016-06-01

    Multi-target drugs, such as the cocktail therapy used for treating AIDS, often show stronger efficacy than single-target drugs in treating complicated diseases. This review will focus on clausenamide (clau), a small molecule compound originally isolated from the traditional Chinese herbal medicine, Clausenalansium. The finding of four chiral centers in clau molecules predicted the presence of 16 clau enantiomers, including (-)-clau and (+)-clau. All of the predicted enantiomers have been successfully synthesized via innovative chemical approaches, and pharmacological studies have demonstrated (-)-clau as a eutomer and (+)-clau as a distomer in improving cognitive function in both normal physiological and pathological conditions. Mechanistically, the nootropic effect of (-)-clau is mediated by its multi-target actions, which include mild elevation of intracellular Ca(2+) concentrations, modulation of the cholinergic system, regulation of synaptic plasticity, and activation of cellular and molecular signaling pathways involved in learning and memory. Furthermore, (-)-clau suppresses the pathogenesis of Alzheimer's disease by inhibiting multiple etiological processes: (1) beta amyloid protein-induced intracellular Ca(2+) overload and apoptosis and (2) tau hyperphosphorylation and neurodegeneration. In conclusion, the nature of the multi-target actions of (-)-clau substantiates it as a promising chiral drug candidate for enhancing human cognition in normal conditions and treating memory impairment in neurodegenerative diseases. PMID:26812265

  18. Sense antisense DNA strand?

    PubMed

    Boldogkói, Z; Kaliman, A V; Murvai, J; Fodor, I

    1994-01-01

    Recent evidence indicates that alphaherpesviruses express latency associated transcripts (LATs) from the antisense strand of immediate-early (IE) genes of the viral genome. It has been suggested that LATs containing extended open reading frames (ORFs), might be translated into (a) protein product(s). We found that a salient feature of some herpesvirus DNAs is a high GC preference at the third codon positions. The consequence of this feature is that the probability of a stop-codon appearing at two of the six reading frames of the DNA strand is very low. Therefore, the presence of an extended ORF does not necessarily mean that it is relevant to real translation. PMID:7810418

  19. 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

  20. Antisense-mediated exon inclusion

    PubMed Central

    Hua, Yimin; Krainer, Adrian R

    2012-01-01

    Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy. PMID:22454070

  1. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD).

    PubMed

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA). PMID:21686247

  2. 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

  3. 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

  4. Multi-Target Stool DNA Test: Is the Future Here?

    PubMed

    Sweetser, Seth; Ahlquist, David A

    2016-06-01

    Colorectal cancer (CRC) screening reduces CRC incidence and mortality and is widely recommended. However, despite these demonstrated benefits, a large percentage of the population remains unscreened. The multi-target stool DNA (MT-sDNA) test is a new, non-invasive option for CRC screening that has a high accuracy rate in detection of colorectal neoplasia and offers great opportunity to enhance screening uptake. This review provides the current state of the art knowledge about the use of MT-sDNA in CRC screening. PMID:27165404

  5. 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.

  6. 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

  7. 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.

  8. Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes.

    PubMed

    Armstrong, Philip M; Prince, Nicholanna; Andreadis, Theodore G

    2012-10-01

    Disease outbreaks caused by eastern equine encephalitis virus (EEEV; Togaviridae, Alphavirus) may be prevented by implementing effective surveillance and intervention strategies directed against the mosquito vector. Methods for EEEV detection in mosquitoes include a real-time reverse transcriptase PCR technique (TaqMan assay), but we report its failure to detect variants isolated in Connecticut in 2011, due to a single base-pair mismatch in the probe-binding site. To improve the molecular detection of EEEV, we developed a multi-target TaqMan assay by adding a second primer/probe set to provide redundant targets for EEEV detection. The multi-target TaqMan assay had similar performance characteristics to the conventional assay, but also detected newly-evolving strains of EEEV. The approach described here increases the reliability of the TaqMan assay by creating back-up targets for virus detection without sacrificing sensitivity or specificity. PMID:22835151

  9. On 'polypharmacy' and multi-target agents, complementary strategies for improving the treatment of depression: a comparative appraisal.

    PubMed

    Millan, Mark J

    2014-07-01

    Major depression is a heterogeneous disorder, both in terms of symptoms, ranging from anhedonia to cognitive impairment, and in terms of pathogenesis, with many interacting genetic, epigenetic, developmental and environmental causes. Accordingly, it seems unlikely that depressive states could be fully controlled by a drug possessing one discrete mechanism of action and, in the wake of disappointing results with several classes of highly selective agent, multi-modal treatment concepts are attracting attention. As concerns pharmacotherapy, there are essentially two core strategies. First, multi-target antidepressants that act via two or more complementary mechanisms and, second, polypharmacy, which refers to co-administration of two distinct drugs, usually in separate pills. Both multi-target agents and polypharmacy ideally couple a therapeutically unexploited action to a clinically established mechanism in order to enhance efficacy, moderate side-effects, accelerate onset of action and treat a broader range of symptoms. The melatonin MT1/MT2 agonist and 5-HT(2C) antagonist, agomelatine, which is effective in the short- and long-term treatment of depression, exemplifies the former approach, while evidence-based polypharmacy is illustrated by the adjunctive use of second-generation antipsychotics with serotonin reuptake inhibitors for treatment of resistant depression. Histone acetylation and methylation, ghrelin signalling, inflammatory modulators, metabotropic glutamate-7 receptors and trace amine-associated-1 receptors comprise attractive substrates for new multi-target and polypharmaceutical strategies. The present article outlines the rationale underpinning multi-modal approaches for treating depression, and critically compares and contrasts the pros and cons of established and potentially novel multi-target vs. polypharmaceutical treatments. On balance, the former appear the most promising for the elaboration, development and clinical implementation of

  10. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo

    NASA Astrophysics Data System (ADS)

    Simons, Michael; Edelman, Elazer R.; Dekeyser, Jean-Luc; Langer, Robert; Rosenberg, Robert D.

    1992-09-01

    SYNTHETIC antisense oligonucleotides have been used to dissect gene function in vitro. Technical difficulties prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense c-myb oligonu-cleotide to suppress intimal accumulation of rat carotid arterial smooth muscle cells. Our results suggest that antisense oligonucleotides can be used to define the in vivo biological role of specific macromolecules in the blood vessel wall and could potentially serve as a new class of therapeutic agents for cardiovascular disorders.

  11. 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

  12. Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

    PubMed

    Bruin, G J; Börnsen, K O; Hüsken, D; Gassmann, E; Widmer, H M; Paulus, A

    1995-08-11

    The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented. PMID:7581844

  13. 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

  14. 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

  15. Pharmaceutical prerequisites for a multi-target therapy.

    PubMed

    Kroll, U; Cordes, C

    2006-01-01

    The quality of a phytomedicine is defined by the quality of the herbal drug, the manufacturing of the drug preparations and the properties of the finished product, taking into account the special requirements of the individual herbal species in accordance with Good Manufacturing Practice (GMP) standards [2003. Medicinal Products for Human and Veterinary Use. Eudralex, vol. 4 (2003/94/EC)]. The quality control of the complete process is based on pharmacognostic methods, characteristic fingerprint chromatograms, defined amounts of marker substances, physicochemical characteristics and microbiological monitoring. For a herbal multi-component preparation used in multi-target therapy, these pharmaceutical prerequisites have to be ensured for all components and for their combination, as is exemplified by Iberogast((R)) (STW 5) a fixed combination of hydroethanolic extracts of bitter candytuft (Iberis amara), angelica root (Angelicae radix), milk thistle fruit (Silybi mariani fructus), celandine herb (Chelidonii herba), caraway fruit (Carvi fructus), liquorice root (Liquiritiae radix), peppermint herb (Menthae piperitae folium), balm leaf (Melissae folium) and chamomile flower (Matricariae flos) using in the therapy of gastrointestinal complaints (Rösch et al., 2006). The prerequisites for the quality of each of its components according to actual standards are at first the cultivation of the plant material according to the Guidelines for Good Agricultural Practice (GAP) conditions of Medicinal and Aromatic Plants [1998. Z. Arzn. Gew. Pfl. 3, 166-178] to yield a defined raw material of high quality. Characteristic compounds of the extracts had to be identified and different analytical methods such as HPLC, with low coefficients of variation had to be developed to analyze each of the standardized ethanolic extracts and the finished product. At the example of the extract of I. amara these necessary investigations are described. The variability of the plant material in its

  16. Multi-target siRNA: Therapeutic Strategy for Hepatocellular Carcinoma

    PubMed Central

    Li, Tiejun; Xue, Yuwen; Wang, Guilan; Gu, Tingting; Li, Yunlong; Zhu, York Yuanyuan; Chen, Li

    2016-01-01

    Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments. PMID:27390607

  17. 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.

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

    PubMed Central

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

    1991-01-01

    We 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-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- 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- permeability. Images PMID:1715578

  19. 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. PMID:26427454

  20. 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.

  1. 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

  2. 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.

  3. 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

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

    PubMed

    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

  5. 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

  6. 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

  7. 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

  8. 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

  9. Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

    PubMed

    Tyler-McMahon, B M; Stewart, J A; Jackson, J; Bitner, M D; Fauq, A; McCormick, D J; Richelson, E

    2001-10-01

    The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter. PMID:11543728

  10. 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.

  11. Classification of compounds with distinct or overlapping multi-target activities and diverse molecular mechanisms using emerging chemical patterns.

    PubMed

    Namasivayam, Vigneshwaran; Hu, Ye; Balfer, Jenny; Bajorath, Jürgen

    2013-06-24

    The emerging chemical patterns (ECP) approach has been introduced for compound classification. Thus far, only very few ECP applications have been reported. Here, we further investigate the ECP methodology by studying complex classification problems. The analysis involves multi-target data sets with systematically organized subsets of compounds having distinct or overlapping target activities and, in addition, data sets containing classes of specifically active compounds with different mechanism-of-action. In systematic classification trials focusing on individual compound subsets or mechanistic classes, ECP calculations utilizing numerical descriptors achieve moderate to high sensitivity, dependent on the data set, and consistently high specificity. Accurate ECP predictions are already obtained on the basis of very small learning sets with only three positive training instances, which distinguishes the ECP approach from many other machine learning techniques. PMID:23692475

  12. 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

  13. 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

  14. Does everything now make (anti)sense?

    PubMed

    Timmons, J A; Good, L

    2006-12-01

    The data generated by the FANTOM (Functional Annotation of Mouse) consortium, Compugen and Affymetrix have collectively provided evidence that most of the mammalian genomes are actively transcribed. The emergence of an antisense RNA world brings new practical complexities to the study and detection of gene expression. However, we also need to address the fundamental questions regarding the functional importance of these molecules. In this brief paper, we focus on non-coding natural antisense transcription, as it appears to be a potentially powerful mechanism for extending the complexity of the protein coding genome, which is currently unable to explain inter-species diversification. PMID:17073772

  15. 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

  16. 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.

  17. Gold nanoparticle mediated laser transfection for high-throughput antisense applications

    NASA Astrophysics Data System (ADS)

    Kalies, S.; Heinemann, D.; Schomaker, M.; Birr, T.; Ripken, T.; Meyer, H.

    2013-06-01

    The delivery of antisense structures, like siRNA, is beneficial for new therapeutic approaches in regenerative sciences. Optical transfection techniques enable high spatial control combined with minimal invasive treatment of cells due to the use of short laser pulses. However, single cell laser transfection by a tightly focused laser beam, for example femtosecond laser transfection, has the major drawback of low throughput. Compared to this, high-throughput in laser transfection is possible by applying gold nanoparticles irradiated by a weakly focused laser beam scanning over the cell sample. Herein, we show the delivery of antisense molecules and demonstrate the minimal cytotoxicity of a method called gold nanoparticle mediated (GNOME) laser transfection. A 532 nm microchip laser in conjugation with 200 nm gold nanoparticles at a concentration of 0.5 μg/cm2 is used. In addition to antisense molecules, the uptake of dextrans of several sizes is analyzed.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    PubMed

    Gilmartin, Daniel J; Soon, Allyson; Thrasivoulou, Christopher; Phillips, Anthony R J; Jayasinghe, Suwan N; Becker, David L

    2016-07-01

    Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed. PMID:27253638

  7. 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

  8. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    PubMed

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  9. Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis.

    PubMed

    Mizu, Masami; Koumoto, Kazuya; Anada, Takahisa; Sakurai, Kazuo; Shinkai, Seiji

    2004-07-01

    Schizophyllan is a beta-(1-->3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cell-free) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect. PMID:14967546

  10. Modulating antibiotic activity towards respiratory bacterial pathogens by co-medications: a multi-target approach.

    PubMed

    Vandevelde, Nathalie M; Tulkens, Paul M; Van Bambeke, Françoise

    2016-07-01

    Non-antibiotic drugs can modulate bacterial physiology and/or antibiotic activity, opening perspectives for innovative therapeutic strategies. Focusing on respiratory pathogens and considering in vitro, in vivo, and clinical data, here we examine the effect of these drugs on the expression of resistance mechanisms, biofilm formation, and intracellular survival, as well as their influence on the activity of antibiotics on bacteria. Beyond the description of the effects observed, we also comment on concentrations that are active and discuss the mechanisms of drug-drug or drug-target interactions. This discussion should be helpful in defining useful targets for adjuvant therapy and establishing the corresponding pharmacophores for further drug fine-tuning. PMID:27094105

  11. Clinical Response of Metastatic Breast Cancer to Multi-targeted Therapeutic Approach: A Single Case Report

    PubMed Central

    Meiners, Christian

    2011-01-01

    The present article describes the ongoing (partial) remission of a female patient (41 years old) from estrogen receptor (ER)-positive/progesterone receptor (PR)-negative metastatic breast cancer in response to a combination treatment directed towards the revitalization of the mitochondrial respiratory chain (oxidative phosphorylation), the suppression of NF-kappaB as a factor triggering the inflammatory response, and chemotherapy with capecitabine. The reduction of tumor mass was evidenced by a continuing decline of CA15-3 and CEA tumor marker serum levels and 18FDG-PET-CT plus magnetic resonance (MR) imaging. It is concluded that such combination treatment might be a useful option for treating already formed metastases and for providing protection against the formation of metastases in ER positive breast cancer. The findings need to be corroborated by clinical trials. Whether similar results can be expected for other malignant tumor phenotypes relying on glycolysis as the main energy source remains to be elucidated. PMID:24212668

  12. The landscape of antisense gene expression in human cancers.

    PubMed

    Balbin, O Alejandro; Malik, Rohit; Dhanasekaran, Saravana M; Prensner, John R; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G; Nesvizhskii, Alexey I; Chinnaiyan, Arul M

    2015-07-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  13. 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.

  14. The landscape of antisense gene expression in human cancers

    PubMed Central

    Balbin, O. Alejandro; Malik, Rohit; Dhanasekaran, Saravana M.; Prensner, John R.; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G.; Nesvizhskii, Alexey I.; Chinnaiyan, Arul M.

    2015-01-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts’ (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  15. 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

  16. 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. PMID:26307001

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    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.

  18. 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

  19. 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

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

    PubMed Central

    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

  1. 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

  2. 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

  3. 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. PMID:27134306

  4. Transfection of normal primary human skeletal myoblasts with p21 and p57 antisense oligonucleotides to improve their proliferation: a first step towards an alternative molecular therapy approach of Duchenne muscular dystrophy.

    PubMed

    Endesfelder, Stefanie; Bucher, Sabine; Kliche, Alexander; Reszka, Regina; Speer, Astrid

    2003-06-01

    Duchenne muscular dystrophy (DMD), caused by the absence of dystrophin, is associated with decreased muscle cell proliferation. An increased p21 mRNA level in DMD patients may be involved in the process. In this context we are interested to improve the proliferation of primary human skeletal muscle cells (SkMC) by a reduction in the cell cycle proteins p21 and p57 using the appropriate antisense oligonucleotides (ASO). Therefore a transfection procedure needs to be optimized in which the oligonucleotide enters the SkMC with a minimal loss of cell vitality and high efficiency. Three different formulations, Effectene, DAC40, and SuperFect, were compared. Proliferation was analyzed comparing cells transfected with p21 and/or p57 ASO vs. cells transfected with scrambled ASO using a bromodeoxyuridine assay. Under optimal conditions (a mixture of 0.25 microg ASO, 5 microl Effectene, 0.8 microl enhancer) SkMC transfected with p21 ASO reveal an average increase in cell proliferation of 32.5+/-11% after 24 h. p57 ASO shows the same effect, but concomitant transfection of p21 and p57 does not enhance it. A cell vitality of 78+/-14% after 24 h was determined by the MTT test. SkMC transfected with DAC40 reveal a maximal increase in proliferation of 38+/-7% after 48 h and show a vitality of 65+/-8%. In contrast to both these formulations, SuperFect was found to be highly toxic for SkMC, with more than 70% dead cells after 24 h. The increase in proliferation, the functional biological effect of p21 ASO, is well correlated with a decrease in p21 detected by western blot analysis of 31.6% for Effectene. Transfection efficiency was measured directly by FACS analysis using FITC-labeled ASO and data showing ASO internalization in 75.8+/-11.2% of the cell population for Effectene and 74.4+/-6.6% cells for DAC40. Taken together transient transfection of p21 or p57 ASO into primary human SkMC using Effectene significantly improves their proliferation compared to transfection with

  5. 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. PMID:26483200

  6. Multi-target screening of biological samples using LC-MS/MS: focus on chromatographic innovations.

    PubMed

    Kohler, Isabelle; Guillarme, Davy

    2014-05-01

    Multi-target screening of biological fluids is a key tool in clinical and forensic toxicology. A complete toxicological analysis encompasses the sample preparation, the chromatographic separation and the detection. The present review briefly covers the new trends in sample preparation and detection and mainly focuses on the chromatographic stage, since a lot of technical improvements have been proposed over the last years. Among them, columns packed with sub-2 μm fully porous particles and sub-3 μm core-shell particles allow for significant improvements of resolution and higher throughput. Even if reversed-phase LC remains the most widely used chromatographic mode for toxicological screening, hydrophilic interaction chromatography and supercritical fluid chromatography appear as promising alternatives for attaining orthogonal selectivity, retention of polar compounds, and enhanced MS sensitivity. PMID:24946925

  7. 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

  8. [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

  9. 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.

  10. Multi-Target Strategy and Experimental Studies of Traditional Chinese Medicine for Alzheimer's Disease Therapy.

    PubMed

    Li, Lin; Zhang, Lan; Yang, Cui-cui

    2016-01-01

    Alzheimer's disease (AD) is a multifactorial complex disease. The pathogenesis of AD is very complicated, and involves the β-amyloid (Aβ) cascade, tau hyperphosphorylation, neuroinflammation, oxidative stress, mitochondrial dysfunction, reduced levels of neurotrophic factors, and damage and loss of synapses as well as cholinergic neurons. The multi-target characteristics of traditional Chinese medicine (TCM) may be advantageous over single-target drugs in the treatment of complex diseases. These drugs have therefore attracted more attention in the research and development of AD therapies. This review describes advances made in experimental studies of TCM for AD treatment. It discusses research, from our group and other laboratories, on TCM compound drugs (Shenwu capsule) and approximately 10 Chinese medicinal herb extracts (tetrahydroxystilbene glucoside, epimedium flavonoid, icariin, cornel iridoid glycoside, ginsenoside, puerarin, clausenamide, huperzine A, and timosaponins). PMID:26268330

  11. 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

  12. [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

  13. 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

  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. 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

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

    PubMed Central

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

    2014-01-01

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

  17. 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

  18. Regulation of the NPT gene by a naturally occurring antisense transcript.

    PubMed

    Werner, Andreas; Preston-Fayers, Keziah; Dehmelt, Leif; Nalbant, Perihan

    2002-01-01

    Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice. PMID:12139410

  19. [Treatment with antisense oligonucleotides in Duchenne's disease].

    PubMed

    Pascual-Pascual, Samuel I

    2012-05-21

    In this paper I review the results of the treatments directed to modify the mRNA of dystrophin with the goal of converting the severe Duchenne type to the milder Becker muscular dystrophy. Antisense oligomers potential to modify Duchenne muscular dystrophy (DMD) gene expression and therapeutic strategies to induce ribosomal read-through of nonsense mutations (PTC124) are described. They are an important advance in the treatment of DMD, so far unspecific. Significant expression of new dystrophin is observed in biopsies of peripheral muscle, although the functional improvement is not so encouraging. New modification of chemistries are expected to improve the liberation, broad distribution in muscles, as well as their efficacy and safety enough to allow a positive chronic treatment of DMD. PMID:22605630

  20. 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

  1. 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.

  2. Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

    PubMed Central

    2015-01-01

    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. PMID:25198246

  3. 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.

  4. Antisense RNA-based High-Throughput Screen System for Directed Evolution of Quorum Quenching Enzymes.

    PubMed

    Han, Sang-Soo; Park, Won-Ji; Kim, Hak-Sung; Kim, Geun-Joong

    2015-11-20

    Quorum quenching (QQ) enzymes, which disrupt the quorum sensing signaling process, have attracted considerable attention as new antimicrobial agents. However, their low catalytic efficiency for quorum sensing molecules remains a challenge. Herein, we present an antisense RNA-based high-throughput screen system for directed evolution of a quorum quenching enzyme. The screening system was constructed by incorporating an antisense RNA (RyhB) into a synthetic module to quantitatively regulate the expression of a reporter gene fused with a sense RNA (sodB). To control the expression of a reporter gene in response to the catalytic activity of a quorum quenching enzyme, the region of interaction and mode between a pair of antisense (RyhB) and sense (sodB) RNAs was designed and optimized through the prediction of the secondary structure of the RNA pair. The screening system constructed was shown to lead to a significant reduction in the false-positive rate (average 42%) in the screening of N-acyl-homoserine lactonase (AiiA) with increased catalytic activity, resulting in a true-positive frequency of up to 76%. The utility and efficiency of the screening system were demonstrated by selecting an AiiA with 31-fold higher catalytic efficiency than the wild-type in three rounds of directed evolution. The present approach can be widely used for the screening of quorum quenching enzymes with the desired catalytic property, as well as for a synthetic network for a stringent regulation of the gene expression. PMID:26366664

  5. Bimodal expression of PHO84 is modulated by early termination of antisense transcription

    PubMed Central

    Castelnuovo, Manuele; Rahman, Samir; Guffanti, Elisa; Infantino, Valentina; Stutz, Françoise; Zenklusen, Daniel

    2016-01-01

    Many S. cerevisiae genes encode antisense transcripts some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 antisense RNA acts as a bimodal switch, where continuous low frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, loss of Rrp6, rather than stabilizing PHO84 antisense RNA, promotes antisense elongation by reducing its early transcription termination by Nrd1-Nab3-Sen1. These observations suggest that PHO84 silencing results from constant low frequency antisense transcription through the promoter rather than its static accumulation at the repressed gene. PMID:23770821

  6. 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

  7. 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.

  8. ATP as a multi-target danger signal in the brain

    PubMed Central

    Rodrigues, Ricardo J.; Tomé, Angelo R.; Cunha, Rodrigo A.

    2015-01-01

    ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R) as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR), which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection. PMID:25972780

  9. 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 shouldnt 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.

  10. 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 shouldnt 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.

  11. UPA-sensitive ACPP-conjugated nanoparticles for multi-targeting therapy of brain glioma.

    PubMed

    Zhang, Bo; Zhang, Yujie; Liao, Ziwei; Jiang, Ting; Zhao, Jingjing; Tuo, Yanyan; She, Xiaojian; Shen, Shun; Chen, Jun; Zhang, Qizhi; Jiang, Xinguo; Hu, Yu; Pang, Zhiqing

    2015-01-01

    Now it is well evidenced that tumor growth is a comprehensive result of multiple pathways, and glioma parenchyma cells and stroma cells are closely associated and mutually compensatory. Therefore, drug delivery strategies targeting both of them simultaneously might obtain more promising therapeutic benefits. In the present study, we developed a multi-targeting drug delivery system modified with uPA-activated cell-penetrating peptide (ACPP) for the treatment of brain glioma (ANP). In vitro experiments demonstrated nanoparticles (NP) decorated with cell-penetrating peptide (CPP) or ACPP could significantly improve nanoparticles uptake by C6 glioma cells and nanoparticles penetration into glioma spheroids as compared with traditional NP and thus enhanced the therapeutic effects of its payload when paclitaxel (PTX) was loaded. In vivo imaging experiment revealed that ANP accumulated more specifically in brain glioma site than NP decorated with or without CPP. Brain slides further showed that ACPP contributed to more nanoparticles accumulation in glioma site, and ANP could co-localize not only with glioma parenchyma cells, but also with stroma cells including neo-vascular cells and tumor associated macrophages. The pharmacodynamics results demonstrated ACPP could significantly improve the therapeutic benefits of nanoparticles by significantly prolonging the survival time of glioma bearing mice. In conclusion, the results suggested that nanoparticles modified with uPA-sensitive ACPP could reach multiple types of cells in glioma tissues and provide a novel strategy for glioma targeted therapy. PMID:25443789

  12. Crawling and walking infants encounter objects differently in a multi-target environment.

    PubMed

    Dosso, Jill A; Boudreau, J Paul

    2014-10-01

    From birth, infants move their bodies in order to obtain information and stimulation from their environment. Exploratory movements are important for the development of an infant's understanding of the world and are well established as being key to cognitive advances. Newly acquired motor skills increase the potential actions available to the infant. However, the way that infants employ potential actions in environments with multiple potential targets is undescribed. The current work investigated the target object selections of infants across a range of self-produced locomotor experience (11- to 14-month-old crawlers and walkers). Infants repeatedly accessed objects among pairs of objects differing in both distance and preference status, some requiring locomotion. Overall, their object actions were found to be sensitive to object preference status; however, the role of object distance in shaping object encounters was moderated by movement status. Crawlers' actions appeared opportunistic and were biased towards nearby objects while walkers' actions appeared intentional and were independent of object position. Moreover, walkers' movements favoured preferred objects more strongly for children with higher levels of self-produced locomotion experience. The multi-target experimental situation used in this work parallels conditions faced by foraging organisms, and infants' behaviours were discussed with respect to optimal foraging theory. There is a complex interplay between infants' agency, locomotor experience, and environment in shaping their motor actions. Infants' movements, in turn, determine the information and experiences offered to infants by their micro-environment. PMID:24888534

  13. Molecular Investigations of Protriptyline as a Multi-Target Directed Ligand in Alzheimer's Disease

    PubMed Central

    Bansode, Sneha B.; Jana, Asis K.; Batkulwar, Kedar B.; Warkad, Shrikant D.; Joshi, Rakesh S.; Sengupta, Neelanjana; Kulkarni, Mahesh J.

    2014-01-01

    Alzheimer's disease (AD) is a complex neurodegenerative disorder involving multiple cellular and molecular processes. The discovery of drug molecules capable of targeting multiple factors involved in AD pathogenesis would greatly facilitate in improving therapeutic strategies. The repositioning of existing non-toxic drugs could dramatically reduce the time and costs involved in developmental and clinical trial stages. In this study, preliminary screening of 140 FDA approved nervous system drugs by docking suggested the viability of the tricyclic group of antidepressants against three major AD targets, viz. Acetylcholinesterase (AChE), β-secretase (BACE-1), and amyloid β (Aβ) aggregation, with one member, protriptyline, showing highest inhibitory activity. Detailed biophysical assays, together with isothermal calorimetry, fluorescence quenching experiments, kinetic studies and atomic force microscopy established the strong inhibitory activity of protriptyline against all three major targets. The molecular basis of inhibition was supported with comprehensive molecular dynamics simulations. Further, the drug inhibited glycation induced amyloid aggregation, another important causal factor in AD progression. This study has led to the discovery of protriptyline as a potent multi target directed ligand and established its viability as a promising candidate for AD treatment. PMID:25141174

  14. 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

  15. ATP as a multi-target danger signal in the brain.

    PubMed

    Rodrigues, Ricardo J; Tomé, Angelo R; Cunha, Rodrigo A

    2015-01-01

    ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R) as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR), which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection. PMID:25972780

  16. 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.

  17. 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

  18. 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

  19. Natural antisense transcription from a comparative perspective.

    PubMed

    Piatek, Monica J; Henderson, Victoria; Zynad, Hany S; Werner, Andreas

    2016-08-01

    Natural antisense transcripts (NATs) can interfere with the expression of complementary sense transcripts with exquisite specificity. We have previously cloned NATs of Slc34a loci (encoding Na-phosphate transporters) from fish and mouse. Here we report the cloning of a human SLC34A1-related NAT that represents an alternatively spliced PFN3 transcript (Profilin3). The transcript is predominantly expressed in testis. Phylogenetic comparison suggests two distinct mechanisms producing Slc34a-related NATs: Alternative splicing of a transcript from a protein coding downstream gene (Pfn3, human/mouse) and transcription from the bi-directional promoter (Rbpja, zebrafish). Expression analysis suggested independent regulation of the complementary Slc34a mRNAs. Analysis of randomly selected bi-directionally transcribed human/mouse loci revealed limited phylogenetic conservation and independent regulation of NATs. They were reduced on X chromosomes and clustered in regions that escape inactivation. Locus structure and expression pattern suggest a NATs-associated regulatory mechanisms in testis unrelated to the physiological role of the sense transcript encoded protein. PMID:27241791

  20. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    PubMed

    Popplewell, Linda J; Malerba, Alberto; Dickson, George

    2012-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene. PMID:22454060

  1. Splice-switching antisense oligonucleotides as therapeutic drugs.

    PubMed

    Havens, Mallory A; Hastings, Michelle L

    2016-08-19

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

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

    PubMed Central

    Chery, Jessica; Näär, Anders

    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

  3. Selenium induces a multi-targeted cell death process in addition to ROS formation.

    PubMed

    Wallenberg, Marita; Misra, Sougat; Wasik, Agata M; Marzano, Cristina; Björnstedt, Mikael; Gandin, Valentina; Fernandes, Aristi P

    2014-04-01

    Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi-target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium-mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno-DL-cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC50 5 μM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis-like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis-like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno-DL-cystine treatment at an IC50 concentration of 100 μM induced morphologically two distinct different types of cell death, one with apoptosis-like phenotype, while the other was reminiscent of paraptosis-like cell death, characterized by induction of unfolded protein response, ER-stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species-specific usage of selenium in the treatment of cancers. PMID:24400844

  4. 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.

  5. On primordial sense-antisense coding

    PubMed Central

    Rodin, Andrei S.; Rodin, Sergei N.; Carter, Charles W.

    2010-01-01

    The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These twenty enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sence-antisence (SAS) coding (Rodin and Ohno, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achylia klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in (Williams et al., 2009), this could indeed be a “Rosetta stone” (eroded somewhat, though) gene for the SAS origin of the two aaRS classes (Carter and Duax, 2002). PMID:19956936

  6. 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

  7. A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

    PubMed Central

    Dominov, Janice A; Uyan, Özgün; Sapp, Peter C; McKenna-Yasek, Diane; Nallamilli, Babi R R; Hegde, Madhuri; Brown, Robert H

    2014-01-01

    Objective Mutations in dysferlin (DYSF), a Ca2+-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches. Methods We sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences. Results We identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full-length DYSF mRNA, and increased DYSF protein expression. Interpretation This is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON-mediated exon-skipping restores production of normal, full-length DYSF in patients’ cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles. PMID:25493284

  8. 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.

  9. Inhibition of dengue virus by novel, modified antisense oligonucleotides.

    PubMed Central

    Raviprakash, K; Liu, K; Matteucci, M; Wagner, R; Riffenburgh, R; Carl, M

    1995-01-01

    Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex. PMID:7983769

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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...

  16. 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

  17. 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

  18. 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

  19. Open reading frames provide a rich pool of potential natural antisense transcripts in fungal genomes.

    PubMed

    Steigele, Stephan; Nieselt, Kay

    2005-01-01

    Natural antisense transcripts are reported from all kingdoms of life and several recent reports of genomewide screens indicate that they are widely distributed. These transcripts seem to be involved in various biological functions and may govern the expression of their respective sense partner. Very little, however, is known about the degree of evolutionary conservation of antisense transcripts. Furthermore, none of the earlier analyses has studied whether antisense relationships are solely dual or involved in more complex relationships. Here we present a systematic screen for cis- and trans-located antisense transcripts based on open reading frames (ORFs) from five fungal species. The relative number of ORFs involved in antisense relationships varies greatly between the five species. In addition, other significant differences are found between the species, such as the mean length of the antisense region. The majority of trans-located antisense transcripts is found to be involved in complex relationships, resulting in highly connected networks. The analysis of the degree of evolutionary conservation of antisense transcripts shows that most antisense transcripts have no ortholog in any other species. An annotation of antisense transcripts based on Gene Ontology directs to common terms and shows that proteins of genes involved in antisense relationships preferentially localize to the nucleus with common functions in the regulation or maintenance of nucleic acids. PMID:16147987

  20. Antisense-Based Progerin Downregulation in HGPS-Like Patients' Cells.

    PubMed

    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

  1. Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

    PubMed Central

    Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.

    2007-01-01

    Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach. PMID:17684229

  2. 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

  3. 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

  4. 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

  5. In vitro characterization of two novel biodegradable vectors for the delivery of radiolabeled antisense oligonucleotides.

    PubMed

    von Guggenberg, Elisabeth; Shahhosseini, Soraya; Koslowsky, Ingrid; Lavasanifar, Afsaneh; Murray, David; Mercer, John

    2010-12-01

    The development of antisense oligonucleotides suitable for tumor targeting applications is hindered by low stability and bioavailability of oligonucleotides in vivo and by the absence of efficient and safe vectors for oligonucleotide delivery. Stabilization in vivo has been achieved through chemical modification of oligonucleotides by various means, but effective approaches to enhance their intracellular delivery are lacking. This study reports on the characterization in vitro of a fully phosphorothioated 20-mer oligonucleotide, complementary to p21 mRNA, radiolabeled with fluorine-18 using a thiol reactive prosthetic group. The potential of two novel synthetic block copolymers containing grafted polyamines on their hydrophobic blocks for vector-assisted cell delivery was studied in vitro. Extensive cellular uptake studies were performed in human colon carcinoma cell lines with enhanced or deficient p21 expression to evaluate and compare the uptake mechanism of naked and vectorized radiolabeled formulations. Uptake studies with the two novel biodegradable vectors showed a moderate increase in cell uptake of the radiofluorinated antisense oligonucleotide. The two vectors show, however, promising advantages over conventional lipidic vectors regarding their biocompatibility and subcellular distribution. PMID:21204767

  6. 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

  7. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    PubMed

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    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

  8. Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

    PubMed

    Uprety, Bhawana; Kaja, Amala; Ferdoush, Jannatul; Sen, Rwik; Bhaumik, Sukesh R

    2016-01-01

    NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription. PMID:26755557

  9. 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. PMID:27174074

  10. 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

  11. 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

  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. PMID:25812965

  13. 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

  14. Molecular and cellular effects of multi-targeted cyclin-dependent kinase inhibition in myeloma: biological and clinical implications.

    PubMed

    McMillin, Douglas W; Delmore, Jake; Negri, Joseph; Buon, Leutz; Jacobs, Hannah M; Laubach, Jacob; Jakubikova, Jana; Ooi, Melissa; Hayden, Patrick; Schlossman, Robert; Munshi, Nikhil C; Lengauer, Christoph; Richardson, Paul G; Anderson, Kenneth C; Mitsiades, Constantine S

    2011-02-01

    Cell cycle regulators, such as cyclin-dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi-targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP-LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub-μmol/l concentrations, spared non-malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF-1α, IRF4. Bortezomib-treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression-free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi-targeted CDK inhibition in MM. PMID:21223249

  15. Episome-generated N-myc antisense RNA restricts the differentiation potential of primitive neuroectodermal cell lines.

    PubMed Central

    Whitesell, L; Rosolen, A; Neckers, L M

    1991-01-01

    Neuroectodermal tumors of childhood provide a unique opportunity to examine the role of genes potentially regulating neuronal growth and differentiation because many cell lines derived from these tumors are composed of at least two distinct morphologic cell types. These types display variant phenotypic characteristics and spontaneously interconvert, or transdifferentiate, in vitro. The factors that regulate transdifferentiation are unknown. Application of antisense approaches to the transdifferentiation process has allowed us to explore the precise role that N-myc may play in regulating developing systems. We now report construction of an episomally replicating expression vector designed to generate RNA antisense to part of the human N-myc gene. Such a vector is able to specifically inhibit N-myc expression in cell lines carrying both normal and amplified N-myc alleles. Inhibition of N-myc expression blocks transdifferentiation in these lines, with accumulation of cells of an intermediate phenotype. A concomitant decrease in growth rate but not loss of tumorigenicity was observed in the N-myc nonamplified cell line CHP-100. Vector-generated antisense RNA should allow identification of genes specifically regulated by the proto-oncogene N-myc. Images PMID:1996098

  16. Nucleolin antisense oligodeoxynucleotides induce apoptosis and may be used as a potential drug for nasopharyngeal carcinoma therapy.

    PubMed

    Wu, Cheng-Der; Chou, Hung-Wen; Kuo, Yuan-Sung; Lu, Ruei-Min; Hwang, Yu-Chyi; Wu, Han-Chung; Lin, Chin-Tarng

    2012-01-01

    Nucleolin (C23, NCL) mRNA was up-regulated in nasopharyngeal carcinoma (NPC) cells compared to that of normal nasomucosal (NNM) cells using a cDNA microarray approach. The level of nucleolin protein was also up-regulated in 13 NPC cell lines, 30 biopsy specimens and nine other cancer cell lines compared to five NNM cells or normal stromal cells, which were analyzed using immunoblotting or immunohistochemistry. We transfected nucleolin antisense oligodeoxynucleotides (phosphorothioate-modified oligodeoxynucleotides; S-ODNs) into NPC-TW01 cells to knockdown nucleolin expression to evaluate the function of nucleolin in cancer cells. Nucleolin knockdown induced NPC cells but not NNM cells to undergo apoptosis. Furthermore, treatment of NPC-TW01 xenograft tumors with nucleolin antisense oligodeoxynucleotides suppressed the growth of xenograft tumors without obvious side effects. Therefore, we suggest that nucleolin may be a potential cancer therapeutic target and that nucleolin antisense oligodeoxynucleotides may be used as a potential drug for therapy in NPC. PMID:21956494

  17. 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

  18. 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

  19. 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

  20. 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

  1. From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for the treatment of Alzheimer's disease

    PubMed Central

    Tarozzi, Andrea; Bartolini, Manuela; Piazzi, Lorna; Valgimigli, Luca; Amorati, Riccardo; Bolondi, Cecilia; Djemil, Alice; Mancini, Francesca; Andrisano, Vincenza; Rampa, Angela

    2014-01-01

    The development of drugs with different pharmacological properties appears to be an innovative therapeutic approach for Alzheimer's disease. In this article, we describe a simple structural modification of AP2238, a first dual function lead, in particular the introduction of the catechol moiety performed in order to search for multi-target ligands. The new compound AP2469 retains anti-acetylcholinesterase (AChE) and beta-site amyloid precursor protein cleaving enzyme (BACE)1 activities compared to the reference, and is also able to inhibit Aβ42 self-aggregation, Aβ42 oligomer-binding to cell membrane and subsequently reactive oxygen species formation in both neuronal and microglial cells. The ability of AP2469 to interfere with Aβ42 oligomer-binding to neuron and microglial cell membrane gives this molecule both neuroprotective and anti-inflammatory properties. These findings, together with its strong chain-breaking antioxidant performance, make AP2469 a potential drug able to modify the course of the disease. PMID:25505579

  2. Analytic Performance Prediction of Track-to-Track Association with Biased Data in Multi-Sensor Multi-Target Tracking Scenarios

    PubMed Central

    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

  3. 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

  4. 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

  5. Targeting Several CAG Expansion Diseases by a Single Antisense Oligonucleotide

    PubMed Central

    Evers, Melvin M.; Pepers, Barry A.; van Deutekom, Judith C. T.; Mulders, Susan A. M.; den Dunnen, Johan T.; Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.; van Roon-Mom, Willeke M. C.

    2011-01-01

    To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2′-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well. PMID:21909428

  6. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    PubMed

    Evers, Melvin M; Pepers, Barry A; van Deutekom, Judith C T; Mulders, Susan A M; den Dunnen, Johan T; Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B; van Roon-Mom, Willeke M C

    2011-01-01

    To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well. PMID:21909428

  7. Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

    PubMed Central

    Lorenz, Peter; Baker, Brenda F.; Bennett, C. Frank; Spector, David L.

    1998-01-01

    Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides. PMID:9571236

  8. 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.

  9. Design, synthesis, and biological evaluation of benzoselenazole-stilbene hybrids as multi-target-directed anti-cancer agents.

    PubMed

    Yan, Jun; Guo, Yueyan; Wang, Yali; Mao, Fei; Huang, Ling; Li, Xingshu

    2015-05-01

    To identify novel multi-target-directed drug candidates for the treatment of cancer, a series of benzoselenazole-stilbene hybrids were synthesised by combining the pharmacophores of resveratrol and ebselen. The biological assay indicated that all of the hybrids exhibited antiproliferative activities against four human cancer cell lines and demonstrated good TrxR inhibitory activities. The mechanism of cell apoptosis was investigated in G2/M cell cycle arrest induced by compound 6e and the apoptosis of the human liver carcinoma Bel-7402 cell line. The significant increase in intracellular ROS confirmed that compound 6e was capable of causing oxidative stress-induced apoptosis in cancer cells. Our results support the potential of compound 6e as a candidate for further studies examining the development of novel drugs for cancer treatment. PMID:25817772

  10. Multi-target screening mines hesperidin as a multi-potent inhibitor: Implication in Alzheimer's disease therapeutics.

    PubMed

    Chakraborty, Sandipan; Bandyopadhyay, Jaya; Chakraborty, Sourav; Basu, Soumalee

    2016-10-01

    Alzheimer's disease (AD) is the most frequent form of neurodegenerative disorder in elderly people. Involvement of several pathogenic events and their interconnections make this disease a complex disorder. Therefore, designing compounds that can inhibit multiple toxic pathways is the most attractive therapeutic strategy in complex disorders like AD. Here, we have designed a multi-tier screening protocol combining ensemble docking to mine BACE1 inhibitor, as well as 2-D QSAR models for anti-amyloidogenic and antioxidant activities. An in house developed phytochemical library of 200 phytochemicals has been screened through this multi-target procedure which mine hesperidin, a flavanone glycoside commonly found in citrus food items, as a multi-potent phytochemical in AD therapeutics. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of hesperidin to the active site of BACE1 induces a conformational transition of the protein from open to closed form. Hesperidin docks close to the catalytic aspartate residues and orients itself in a way that blocks the cavity opening thereby precluding substrate binding. Hesperidin is a high affinity BACE1 inhibitor and only 500 nM of the compound shows complete inhibition of the enzyme activity. Furthermore, ANS and Thioflavin-T binding assay show that hesperidin completely inhibits the amyloid fibril formation which is further supported by atomic force microscopy. Hesperidin exhibits moderate ABTS(+) radical scavenging assay but strong hydroxyl radical scavenging ability, as evident from DNA nicking assay. Present study demonstrates the applicability of a novel multi-target screening procedure to mine multi-potent agents from natural origin for AD therapeutics. PMID:27068363

  11. Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria.

    PubMed

    Seligmann, Hervé

    2010-07-01

    Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the 'antisense' tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs. PMID:20399828

  12. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model. PMID:22869723

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

    PubMed

    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 (131)I-NGA and (99m)Tc-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, (99m)Tc-3P-RGD2 had the ability to evaluate liver fibrosis and detect tumor lesions. (131)I-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 (99m)Tc-3P-RGD2 and (131)I-NGA is an effective approach to detect liver disease, especially liver fibrosis and liver cancer. PMID:27377130

  15. 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

  16. A riboswitch-regulated antisense RNA in Listeria monocytogenes.

    PubMed

    Mellin, J R; Tiensuu, Teresa; Bécavin, Christophe; Gouin, Edith; Johansson, Jörgen; Cossart, Pascale

    2013-08-01

    Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs. PMID:23878253

  17. Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

    PubMed Central

    Mazet, Muriel; Perozzo, Remo; Bergamini, Christian; Prati, Federica; Fato, Romana; Lenaz, Giorgio; Capranico, Giovanni; Brun, Reto; Bakker, Barbara M.; Michels, Paul A. M.; Scapozza, Leonardo; Bolognesi, Maria Laura; Cavalli, Andrea

    2013-01-01

    Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands. PMID:23350008

  18. 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.

  19. 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

  20. 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…

  1. 3'-modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium.

    PubMed Central

    Hinrichsen, R D; Fraga, D; Reed, M W

    1992-01-01

    The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology. PMID:1528867

  2. Characteristics of Antisense Transcript Promoters and the Regulation of Their Activity

    PubMed Central

    Lin, Shudai; Zhang, Li; Luo, Wen; Zhang, Xiquan

    2015-01-01

    Recently, an increasing number of studies on natural antisense transcripts have been reported, especially regarding their classification, temporal and spatial expression patterns, regulatory functions and mechanisms. It is well established that natural antisense transcripts are produced from the strand opposite to the strand encoding a protein. Despite the pivotal roles of natural antisense transcripts in regulating the expression of target genes, the transcriptional mechanisms initiated by antisense promoters (ASPs) remain unknown. To date, nearly all of the studies conducted on this topic have focused on the ASP of a single gene of interest, whereas no study has systematically analyzed the locations of ASPs in the genome, ASP activity, or factors influencing this activity. This review focuses on elaborating on and summarizing the characteristics of ASPs to extend our knowledge about the mechanisms of antisense transcript initiation. PMID:26703594

  3. 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

  4. 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

  5. Multi-targeted therapy of cancer by omega-3 fatty acids.

    PubMed

    Berquin, Isabelle M; Edwards, Iris J; Chen, Yong Q

    2008-10-01

    Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids necessary for human health. Currently, the Western diet contains a disproportionally high amount of n-6 PUFAs and low amount of n-3 PUFAs, and the resulting high n-6/n-3 ratio is thought to contribute to cardiovascular disease, inflammation, and cancer. Studies in human populations have linked high consumption of fish or fish oil to reduced risk of colon, prostate, and breast cancer, although other studies failed to find a significant association. Nonetheless, the available epidemiological evidence, combined with the demonstrated effects of n-3 PUFAs on cancer in animal and cell culture models, has motivated the development of clinical interventions using n-3 PUFAs in the prevention and treatment of cancer, as well as for nutritional support of cancer patients to reduce weight loss and modulate the immune system. In this review, we discuss the rationale for using long-chain n-3 PUFAs in cancer prevention and treatment and the challenges that such approaches pose in the design of clinical trials. PMID:18479809

  6. Multi-Targeted Mechanisms Underlying the Endothelial Protective Effects of the Diabetic-Safe Sweetener Erythritol

    PubMed Central

    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. PMID:23755276

  7. 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.

  8. 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

  9. Extensive Polycistronism and Antisense Transcription in the Mammalian Hox Clusters

    PubMed Central

    Mainguy, Gaëll; Koster, Jan; Woltering, Joost; Jansen, Hans; Durston, Antony

    2007-01-01

    The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70%) have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp) overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb). This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering. PMID:17406680

  10. 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

  11. 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

  12. 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.

  13. Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase.

    PubMed Central

    Palaparti, A; Li, Y; Anand-Srivastava, M B

    2000-01-01

    Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not

  14. Strand-Specific RNA-Seq Reveals Ordered Patterns of Sense and Antisense Transcription in Bacillus anthracis

    PubMed Central

    Passalacqua, Karla D.; Varadarajan, Anjana; Weist, Charlotte; Ondov, Brian D.; Byrd, Benjamin; Read, Timothy D.; Bergman, Nicholas H.

    2012-01-01

    Background Although genome-wide transcriptional analysis has been used for many years to study bacterial gene expression, many aspects of the bacterial transcriptome remain undefined. One example is antisense transcription, which has been observed in a number of bacteria, though the function of antisense transcripts, and their distribution across the bacterial genome, is still unclear. Methodology/Principal Findings Single-stranded RNA-seq results revealed a widespread and non-random pattern of antisense transcription covering more than two thirds of the B. anthracis genome. Our analysis revealed a variety of antisense structural patterns, suggesting multiple mechanisms of antisense transcription. The data revealed several instances of sense and antisense expression changes in different growth conditions, suggesting that antisense transcription may play a role in the ways in which B. anthracis responds to its environment. Significantly, genome-wide antisense expression occurred at consistently higher levels on the lagging strand, while the leading strand showed very little antisense activity. Intrasample gene expression comparisons revealed a gene dosage effect in all growth conditions, where genes farthest from the origin showed the lowest overall range of expression for both sense and antisense directed transcription. Additionally, transcription from both strands was verified using a novel strand-specific assay. The variety of structural patterns we observed in antisense transcription suggests multiple mechanisms for this phenomenon, suggesting that some antisense transcription may play a role in regulating the expression of key genes, while some may be due to chromosome replication dynamics and transcriptional noise. Conclusions/Significance Although the variety of structural patterns we observed in antisense transcription suggest multiple mechanisms for antisense expression, our data also clearly indicate that antisense transcription may play a genome-wide role

  15. Synthesis and evaluation of multi-target-directed ligands against Alzheimer's disease based on the fusion of donepezil and ebselen.

    PubMed

    Luo, Zonghua; Sheng, Jianfei; Sun, Yang; Lu, Chuanjun; Yan, Jun; Liu, Anqiu; Luo, Hai-Bin; Huang, Ling; Li, Xingshu

    2013-11-27

    A novel series of compounds obtained by fusing the cholinesterase inhibitor donepezil and the antioxidant ebselen were designed as multi-target-directed ligands against Alzheimer's disease. An in vitro assay showed that some of these molecules did not exhibit highly potent cholinesterase inhibitory activity but did have various other ebselen-related pharmacological effects. Among the molecules, compound 7d, one of the most potent acetylcholinesterase inhibitors (IC50 values of 0.042 μM for Electrophorus electricus acetylcholinesterase and 0.097 μM for human acetylcholinesterase), was found to be a strong butyrylcholinesterase inhibitor (IC50 = 1.586 μM), to possess rapid H2O2 and peroxynitrite scavenging activity and glutathione peroxidase-like activity (ν0 = 123.5 μM min(-1)), and to be a substrate of mammalian TrxR. A toxicity test in mice showed no acute toxicity at doses of up to 2000 mg/kg. According to an in vitro blood-brain barrier model, 7d is able to penetrate the central nervous system. PMID:24160297

  16. 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

  17. Unconstrained underwater multi-target tracking in passive sonar systems using two-stage PF-based technique

    NASA Astrophysics Data System (ADS)

    Georgy, Jacques; Noureldin, Aboelmagd

    2014-03-01

    A robust particle filter (PF)-based multi-target tracking solution for passive sonar systems able to track an unknown time-varying number of multiple targets, while keeping continuous tracks of such targets, is presented in this article. PF is a nonlinear filtering technique that can accommodate arbitrary sensor characteristics, motion dynamics and noise distributions. An enhanced version of PF is employed and is called Mixture PF. The commonly used sampling/importance resampling PF samples from the prior importance density, while Mixture PF samples from both the prior and the observation likelihood. In order to be able to track an unknown time-varying number of multiple targets, two Mixture PFs are used, one for target detection and the other for tracking multiple targets, and a density-based clustering technique is used after the first filter. This article demonstrates the applicability of the proposed technique for the passive problem, which suffers from the lack of measurements and the small detection range of the buoys, especially for weak signals. A contact-level simulation was used to generate different scenarios and the performance of the proposed technique called Clustered-Mixture PF was examined with either bearing measurement only or bearing and Doppler measurements, and it demonstrated its high performance.

  18. 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

  19. 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

  20. 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

  1. Nano-crystalline Ag-PbTe thermoelectric thin films by a multi-target PLD system

    NASA Astrophysics Data System (ADS)

    Cappelli, E.; Bellucci, A.; Medici, L.; Mezzi, A.; Kaciulis, S.; Fumagalli, F.; Di Fonzo, F.; Trucchi, D. M.

    2015-05-01

    It has been evaluated the ability of ArF pulsed laser ablation to grow nano-crystalline thin films of high temperature PbTe thermoelectric material, and to obtain a uniform and controlled Ag blending, through the entire thickness of the film, using a multi-target system in vacuum. The substrate used was a mirror polished technical alumina slab. The increasing atomic percentage of Ag effect on physical-chemical and electronic properties was evaluated in the range 300-575 K. The stoichiometry and the distribution of the Ag component, over the whole thickness of the samples deposited, have been studied by XPS (X-ray photoelectron spectroscopy) and corresponding depth profiles. The crystallographic structure of the film was analyzed by grazing incidence X-ray diffraction (GI-XRD) system. Scherrer analysis for crystallite size shows the presence of nano-structures, of the order of 30-35 nm. Electrical resistivity of the samples, studied by the four point probe method, as a function of increasing Ag content, shows a typical semi-conductor behavior. From conductivity values, carrier concentration and Seebeck parameter determination, the power factor of deposited films was calculated. Both XPS, Hall mobility and Seebeck analysis seem to indicate a limit value to the Ag solubility of the order of 5%, for thin films of ∼200 nm thickness, deposited at 350 °C. These data resulted to be comparable to theoretical evaluation for thin films but order of magnitude lower than the corresponding bulk materials.

  2. 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

  3. 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.

  4. 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. PMID:23922646

  5. 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

  6. 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

  7. 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

  8. Photosynthetic Performance and Fertility Are Repressed in GmAOX2b Antisense Soybean1[OA

    PubMed Central

    Chai, Tsun-Thai; Simmonds, Daina; Day, David A.; Colmer, Timothy D.; Finnegan, Patrick M.

    2010-01-01

    The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function. PMID:20097793

  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. Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

    PubMed

    Rimessi, Paola; Fabris, Marina; Bovolenta, Matteo; Bassi, Elena; Falzarano, Sofia; Gualandi, Francesca; Rapezzi, Claudio; Coccolo, Fabio; Perrone, Daniela; Medici, Alessandro; Ferlini, Alessandra

    2010-09-01

    Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches. PMID:20486769

  13. Antisense oligonucleotide induction of progerin in human myogenic cells.

    PubMed

    Luo, Yue-Bei; Mitrpant, Chalermchai; Adams, Abbie M; Johnsen, Russell D; Fletcher, Sue; Mastaglia, Frank L; Wilton, Steve D

    2014-01-01

    We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate

  14. Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

    PubMed Central

    Canning, Peter; Tan, Li; Chu, Kiki; Lee, Sam W.; Gray, Nathanael S.; Bullock, Alex N.

    2014-01-01

    The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer. PMID:24768818

  15. 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

  16. A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease.

    PubMed

    Takedatsu, Hidetoshi; Mitsuyama, Keiichi; Mochizuki, Shinichi; Kobayashi, Teppei; Sakurai, Kazuo; Takeda, Hiroshi; Fujiyama, Yoshihide; Koyama, Yoshikazu; Nishihira, Jun; Sata, Michio

    2012-06-01

    Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the β-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD. PMID:22334022

  17. A New Therapeutic Approach Using a Schizophyllan-based Drug Delivery System for Inflammatory Bowel Disease

    PubMed Central

    Takedatsu, Hidetoshi; Mitsuyama, Keiichi; Mochizuki, Shinichi; Kobayashi, Teppei; Sakurai, Kazuo; Takeda, Hiroshi; Fujiyama, Yoshihide; Koyama, Yoshikazu; Nishihira, Jun; Sata, Michio

    2012-01-01

    Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the β-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD. PMID:22334022

  18. Combination antisense treatment for destructive exon skipping of myostatin and open reading frame rescue of dystrophin in neonatal mdx mice

    PubMed Central

    Lu-Nguyen, Ngoc B.; Jarmin, Susan A.; Saleh, Amer F.; Popplewell, Linda; Gait, Michael J.; Dickson, George

    2015-01-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  19. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    PubMed

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  20. 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

  1. Antisense-induced messenger depletion corrects a COL6A2 dominant mutation in Ullrich myopathy.

    PubMed

    Gualandi, Francesca; Manzati, Elisa; Sabatelli, Patrizia; Passarelli, Chiara; Bovolenta, Matteo; Pellegrini, Camilla; Perrone, Daniela; Squarzoni, Stefano; Pegoraro, Elena; Bonaldo, Paolo; Ferlini, Alessandra

    2012-12-01

    Collagen VI gene mutations cause Ullrich and Bethlem muscular dystrophies. Pathogenic mutations frequently have a dominant negative effect, with defects in collagen VI chain secretion and assembly. It is agreed that, conversely, collagen VI haploinsufficiency has no pathological consequences. Thus, RNA-targeting approaches aimed at preferentially inactivating the mutated COL6 messenger may represent a promising therapeutic strategy. By in vitro studies we obtained the preferential depletion of the mutated COL6A2 messenger, by targeting a common single-nucleotide polymorphism (SNP), cistronic with a dominant COL6A2 mutation. We used a 2'-O-methyl phosphorothioate (2'OMePS) antisense oligonucleotide covering the SNP within exon 3, which is out of frame. Exon 3 skipping has the effect of depleting the mutated transcript via RNA nonsense-mediated decay, recovering the correct collagen VI secretion and restoring the ability to form an interconnected microfilament network into the extracellular matrix. This novel RNA modulation approach to correcting dominant mutations may represent a therapeutic strategy potentially applicable to a great variety of mutations and diseases. PMID:22992134

  2. 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

  3. 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

  4. 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

  5. An egr-1 (zif268) Antisense Oligodeoxynucleotide Infused Into the Amygdala Disrupts Fear Conditioning

    PubMed Central

    Malkani, Seema; Wallace, Karin J.; Donley, Melanie P.; Rosen, Jeffrey B.

    2004-01-01

    Studies of gene expression following fear conditioning have demonstrated that the inducible transcription factor, egr-1, is increased in the lateral nucleus of the amygdala shortly following fear conditioning. These studies suggest that egr-1 and its protein product Egr-1 in the amygdala are important for learning and memory of fear. To directly test this hypothesis, an egr-1 antisense oligodeoxynucleotide (antisense-ODN) was injected bilaterally into the amygdala prior to contextual fear conditioning. The antisense-ODN reduced Egr-1 protein in the amygdala and interfered with fear conditioning. A 250-pmole dose produced an 11% decrease in Egr-1 protein and reduced long-term memory of fear as measured by freezing in a retention test 24 h after conditioning, but left shock-induced freezing intact. A larger 500-pmole dose produced a 25% reduction in Egr-1 protein and significantly decreased both freezing immediately following conditioning and freezing in the retention test. A nonsense-ODN had no effect on postshock or retention test freezing. In addition, 500 pmole of antisense-ODN infused prior to the retention test in previously trained rats did not reduce freezing, indicating that antisense-ODN did not suppress conditioned fear behavior. Finally, rats infused with 500 pmole of antisense-ODN displayed unconditioned fear to a predator odor, demonstrating that unconditioned freezing was unaffected by the antisense-ODN. The data indicate that the egr-1 antisense-ODN interferes with learning and memory processes of fear without affecting freezing behavior and suggests that the inducible transcription factor Egr-1 within the amygdala plays important functions in long-term learning and memory of fear. PMID:15466317

  6. 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

  7. Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

    PubMed

    Karinaga, Ryouji; Anada, Takahisa; Minari, Jusaku; Mizu, Masami; Koumoto, Kazuya; Fukuda, Junji; Nakazawa, Kohji; Hasegawa, Teruaki; Numata, Munenori; Shinkai, Seiji; Sakurai, Kazuo

    2006-03-01

    Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery. PMID:16174528

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

    PubMed

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

    2015-01-01

    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

  9. A novel antisense long noncoding RNA regulates the expression of MDC1 in bladder cancer

    PubMed Central

    Hua, Qiuhan; Chu, Haiyan; Tong, Na; Yuan, Lin; Qin, Chao; Yin, Changjun; Zhang, Zhengdong; Wang, Meilin

    2015-01-01

    Antisense long noncoding RNAs (lncRNAs) play important roles in regulating the expression of coding genes in post-transcriptional level. However, detailed expression profile of lncRNAs and functions of antisense lncRNAs in bladder cancer remains unclear. To investigate regulation of lncRNAs in bladder cancer and demonstrate their functions, we performed lncRNAs microarray analysis in 3 paired bladder cancer tissues. Further molecular assays were conducted to determine the potential role of identified antisense lncRNA MDC1-AS. As a result, a series of lncRNAs were differentially expressed in bladder cancer tissues in microarray screen. In a larger size of samples validation, we found that the expression levels of MDC1-AS and MDC1 was down-regulated in bladder cancer. After over-expression of MDC1-AS, increased levels of MDC1 were observed in bladder cancer cells. We also found a remarkably inhibitory role of antisense lncRNA MDC1-AS on malignant cell behaviors in bladder cancer cells EJ and T24. Subsequently, knockdown of MDC1 revealed that suppressing role of MDC1-AS was attributed to up-regulation of MDC1. In summary, we have identified a novel antisense lncRNA MDC1-AS, which may participate in bladder cancer through up-regulation of its antisense tumor-suppressing gene MDC1. Further studies should be conducted to demonstrate detailed mechanism of our findings. PMID:25514464

  10. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230