Science.gov

Sample records for induces targeted rewiring

  1. ATR inhibition rewires cellular signaling networks induced by replication stress.

    PubMed

    Wagner, Sebastian A; Oehler, Hannah; Voigt, Andrea; Dalic, Denis; Freiwald, Anja; Serve, Hubert; Beli, Petra

    2016-02-01

    The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling. PMID:26572502

  2. The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

    PubMed Central

    Childers, Delma S.; Raziunaite, Ingrida; Mol Avelar, Gabriela; Mackie, Joanna; Budge, Susan; Stead, David; Gow, Neil A. R.; Lenardon, Megan D.; Ballou, Elizabeth R.; MacCallum, Donna M.; Brown, Alistair J. P.

    2016-01-01

    Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is “Crabtree positive”, displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for

  3. The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence.

    PubMed

    Childers, Delma S; Raziunaite, Ingrida; Mol Avelar, Gabriela; Mackie, Joanna; Budge, Susan; Stead, David; Gow, Neil A R; Lenardon, Megan D; Ballou, Elizabeth R; MacCallum, Donna M; Brown, Alistair J P

    2016-04-01

    Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in

  4. Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression

    PubMed Central

    Zheng, Li; Dai, Huifang; Zhou, Mian; Li, Xiaojin; Liu, Changwei; Guo, Zhigang; Wu, Xiwei; Wu, Jun; Wang, Charles; Zhong, John; Huang, Qin; Garcia-Aguilar, Julio; Pfeifer, Gerd P.; Shen, Binghui

    2012-01-01

    Mutations in genes involved in DNA replication such as FEN1, can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumor progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that formation of polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes, to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumor cells, enabling them to escape replication stress-induced senescence barriers. PMID:22569363

  5. Enriched and Deprived Sensory Experience Induces Structural Changes and Rewires Connectivity during the Postnatal Development of the Brain

    PubMed Central

    Bengoetxea, Harkaitz; Ortuzar, Naiara; Bulnes, Susana; Rico-Barrio, Irantzu; Lafuente, José Vicente; Argandoña, Enrike G.

    2012-01-01

    During postnatal development, sensory experience modulates cortical development, inducing numerous changes in all of the components of the cortex. Most of the cortical changes thus induced occur during the critical period, when the functional and structural properties of cortical neurons are particularly susceptible to alterations. Although the time course for experience-mediated sensory development is specific for each system, postnatal development acts as a whole, and if one cortical area is deprived of its normal sensory inputs during early stages, it will be reorganized by the nondeprived senses in a process of cross-modal plasticity that not only increases performance in the remaining senses when one is deprived, but also rewires the brain allowing the deprived cortex to process inputs from other senses and cortices, maintaining the modular configuration. This paper summarizes our current understanding of sensory systems development, focused specially in the visual system. It delineates sensory enhancement and sensory deprivation effects at both physiological and anatomical levels and describes the use of enriched environment as a tool to rewire loss of brain areas to enhance other active senses. Finally, strategies to apply restorative features in human-deprived senses are studied, discussing the beneficial and detrimental effects of cross-modal plasticity in prostheses and sensory substitution devices implantation. PMID:22848849

  6. A UV–Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring

    PubMed Central

    Srivas, Rohith; Costelloe, Thomas; Carvunis, Anne-Ruxandra; Sarkar, Sovan; Malta, Erik; Sun, Su Ming; Pool, Marijke; Licon, Katherine; van Welsem, Tibor; van Leeuwen, Fred; McHugh, Peter J.; van Attikum, Haico; Ideker, Trey

    2014-01-01

    SUMMARY Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions amongst 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks. PMID:24360959

  7. Bypass rewiring and robustness of complex networks.

    PubMed

    Park, Junsang; Hahn, Sang Geun

    2016-08-01

    A concept of bypass rewiring is introduced, and random bypass rewiring is analytically and numerically investigated with simulations. Our results show that bypass rewiring makes networks robust against removal of nodes including random failures and attacks. In particular, random bypass rewiring connects all nodes except the removed nodes on an even degree infinite network and makes the percolation threshold 0 for arbitrary occupation probabilities. In our example, the even degree network is more robust than the original network with random bypass rewiring, while the original network is more robust than the even degree networks without random bypass. We propose a greedy bypass rewiring algorithm which guarantees the maximum size of the largest component at each step, assuming which node will be removed next is unknown. The simulation result shows that the greedy bypass rewiring algorithm improves the robustness of the autonomous system of the Internet under attacks more than random bypass rewiring. PMID:27627320

  8. Robust criticality of an Ising model on rewired directed networks

    NASA Astrophysics Data System (ADS)

    Lipowski, Adam; Gontarek, Krzysztof; Lipowska, Dorota

    2015-06-01

    We show that preferential rewiring, which is supposed to mimic the behavior of financial agents, changes a directed-network Ising ferromagnet with a single critical point into a model with robust critical behavior. For the nonrewired random graph version, due to a constant number of out-links for each site, we write a simple mean-field-like equation describing the behavior of magnetization; we argue that it is exact and support the claim with extensive Monte Carlo simulations. For the rewired version, this equation is obeyed only at low temperatures. At higher temperatures, rewiring leads to strong heterogeneities, which apparently invalidates mean-field arguments and induces large fluctuations and divergent susceptibility. Such behavior is traced back to the formation of a relatively small core of agents that influence the entire system.

  9. Intraspinal Rewiring of the Corticospinal Tract Requires Target-Derived Brain-Derived Neurotrophic Factor and Compensates Lost Function after Brain Injury

    ERIC Educational Resources Information Center

    Ueno, Masaki; Hayano, Yasufumi; Nakagawa, Hiroshi; Yamashita, Toshihide

    2012-01-01

    Brain injury that results in an initial behavioural deficit is frequently followed by spontaneous recovery. The intrinsic mechanism of this functional recovery has never been fully understood. Here, we show that reorganization of the corticospinal tract induced by target-derived brain-derived neurotrophic factor is crucial for spontaneous recovery…

  10. Kicking Genomic Profiling to the Curb: How Re-wiring the Phosphoproteome Can Explain Treatment Resistance in Glioma.

    PubMed

    Lam, Fred C; Yaffe, Michael B

    2016-04-11

    In this issue of Cancer Cell, Wei et al. (2016) identify adaptive re-wiring of signaling nodes in glioma as major mechanisms of treatment resistance without genome-wide mutations. Targeting these nodes before treatment blocks resistance and underscores the importance of single-cell phosphoproteomics and network re-wiring in predicting cancer treatment responses. PMID:27070697

  11. Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain

    PubMed Central

    Hayashi, Hideaki; Ishikawa, Tatsuya; Shibata, Keisuke; Inada, Hiroyuki; Roh, Seung Eon; Kim, Sang Jeong; Moorhouse, Andrew J.

    2016-01-01

    Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia. PMID:27064281

  12. Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain.

    PubMed

    Kim, Sun Kwang; Hayashi, Hideaki; Ishikawa, Tatsuya; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Youichi; Inada, Hiroyuki; Roh, Seung Eon; Kim, Sang Jeong; Lee, Gihyun; Bae, Hyunsu; Moorhouse, Andrew J; Mikoshiba, Katsuhiko; Fukazawa, Yugo; Koizumi, Schuichi; Nabekura, Junichi

    2016-05-01

    Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia. PMID:27064281

  13. Pneumaplasticity: Rewiring the Human Soul.

    PubMed

    Cherwien, Karen Martinson

    2016-06-01

    We human beings continue to wrestle with our identities as spiritual people, both individually and collectively. This article proposes a synthesized perspective on human spirituality, introducing the concept of pneumaplasticity, which allows us to move beyond the spiritual limits common to several major faith traditions to see how our spiritual selves can adapt and rewire to facilitate meaning-making and continued discernment of our ever-developing senses of identity and meaning. This concept is of particular value to professionals providing spiritual care in any setting. PMID:27281762

  14. Improving Network Transport Efficiency by Edge Rewiring

    NASA Astrophysics Data System (ADS)

    Jiang, Zhong-Yuan; Liang, Man-Gui; Guo, Dong-Chao

    2013-03-01

    Considering the heterogeneous structure of scale-free networks causing low traffic capacity of network, we propose to improve the network transport efficiency by rewiring a fraction of edges for the network. In this paper, six edge rewiring strategies are discussed and extensive simulations on Barabási-Albert (BA) scale-free networks confirm the effectiveness of these strategies. From another perspective, rewiring edges for scale-free networks directly reuse the removed edges under some edge-removal strategies [Z. Liu, M. B. Hu, R. Jiang, W. X. Wang and Q. S. Wu, Phys. Rev. E76 (2007) 037101; G. Q. Zhang, D. Wang and G. J. Li, Phys. Rev. E76 (2007) 017101], and can significantly enhance the traffic capacity of the network at the expense of increasing a little average path length. After the edge rewiring process, the network structure becomes significantly homogeneous. This work is helpful for network design and network performance optimization.

  15. Rapid Mechanically Controlled Rewiring of Neuronal Circuits.

    PubMed

    Magdesian, Margaret H; Lopez-Ayon, G Monserratt; Mori, Megumi; Boudreau, Dominic; Goulet-Hanssens, Alexis; Sanz, Ricardo; Miyahara, Yoichi; Barrett, Christopher J; Fournier, Alyson E; De Koninck, Yves; Grütter, Peter

    2016-01-20

    CNS injury may lead to permanent functional deficits because it is still not possible to regenerate axons over long distances and accurately reconnect them with an appropriate target. Using rat neurons, microtools, and nanotools, we show that new, functional neurites can be created and precisely positioned to directly (re)wire neuronal networks. We show that an adhesive contact made onto an axon or dendrite can be pulled to initiate a new neurite that can be mechanically guided to form new synapses at up to 0.8 mm distance in <1 h. Our findings challenge current understanding of the limits of neuronal growth and have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration. Significance statement: Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Using microtools and nanotools we have developed a new method to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved are ≥60 times faster than previously reported. Our findings have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration after trauma and in neurodegenerative diseases. It also opens the door for the direct wiring of robust brain-machine interfaces as well as for investigations of fundamental aspects of neuronal signal processing and neuronal function. PMID:26791225

  16. Measuring the evolutionary rewiring of biological networks.

    PubMed

    Shou, Chong; Bhardwaj, Nitin; Lam, Hugo Y K; Yan, Koon-Kiu; Kim, Philip M; Snyder, Michael; Gerstein, Mark B

    2011-01-01

    We have accumulated a large amount of biological network data and expect even more to come. Soon, we anticipate being able to compare many different biological networks as we commonly do for molecular sequences. It has long been believed that many of these networks change, or "rewire", at different rates. It is therefore important to develop a framework to quantify the differences between networks in a unified fashion. We developed such a formalism based on analogy to simple models of sequence evolution, and used it to conduct a systematic study of network rewiring on all the currently available biological networks. We found that, similar to sequences, biological networks show a decreased rate of change at large time divergences, because of saturation in potential substitutions. However, different types of biological networks consistently rewire at different rates. Using comparative genomics and proteomics data, we found a consistent ordering of the rewiring rates: transcription regulatory, phosphorylation regulatory, genetic interaction, miRNA regulatory, protein interaction, and metabolic pathway network, from fast to slow. This ordering was found in all comparisons we did of matched networks between organisms. To gain further intuition on network rewiring, we compared our observed rewirings with those obtained from simulation. We also investigated how readily our formalism could be mapped to other network contexts; in particular, we showed how it could be applied to analyze changes in a range of "commonplace" networks such as family trees, co-authorships and linux-kernel function dependencies. PMID:21253555

  17. Global coordination in adaptation to gene rewiring

    PubMed Central

    Murakami, Yoshie; Matsumoto, Yuki; Tsuru, Saburo; Ying, Bei-Wen; Yomo, Tetsuya

    2015-01-01

    Gene rewiring is a common evolutionary phenomenon in nature that may lead to extinction for living organisms. Recent studies on synthetic biology demonstrate that cells can survive genetic rewiring. This survival (adaptation) is often linked to the stochastic expression of rewired genes with random transcriptional changes. However, the probability of adaptation and the underlying common principles are not clear. We performed a systematic survey of an assortment of gene-rewired Escherichia coli strains to address these questions. Three different cell fates, designated good survivors, poor survivors and failures, were observed when the strains starved. Large fluctuations in the expression of the rewired gene were commonly observed with increasing cell size, but these changes were insufficient for adaptation. Cooperative reorganizations in the corresponding operon and genome-wide gene expression largely contributed to the final success. Transcriptome reorganizations that generally showed high-dimensional dynamic changes were restricted within a one-dimensional trajectory for adaptation to gene rewiring, indicating a general path directed toward cellular plasticity for a successful cell fate. This finding of global coordination supports a mechanism of stochastic adaptation and provides novel insights into the design and application of complex genetic or metabolic networks. PMID:25564530

  18. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.

    PubMed

    Tsao, Chen-Yu; Hooshangi, Sara; Wu, Hsuan-Chen; Valdes, James J; Bentley, William E

    2010-05-01

    Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions. PMID:20060924

  19. miRNA-Mediated KHSRP Silencing Rewires Distinct Post-transcriptional Programs during TGF-β-Induced Epithelial-to-Mesenchymal Transition.

    PubMed

    Puppo, Margherita; Bucci, Gabriele; Rossi, Martina; Giovarelli, Matteo; Bordo, Domenico; Moshiri, Arfa; Gorlero, Franco; Gherzi, Roberto; Briata, Paola

    2016-07-26

    Epithelial-to-mesenchymal transition (EMT) confers several traits to cancer cells that are required for malignant progression. Here, we report that miR-27b-3p-mediated silencing of the single-strand RNA binding protein KHSRP is required for transforming growth factor β (TGF-β)-induced EMT in mammary gland cells. Sustained KHSRP expression limits TGF-β-dependent induction of EMT factors and cell migration, whereas its knockdown in untreated cells mimics TGF-β-induced EMT. Genome-wide sequencing analyses revealed that KHSRP controls (1) levels of mature miR-192-5p, a microRNA that targets a group of EMT factors, and (2) alternative splicing of a cohort of pre-mRNAs related to cell adhesion and motility including Cd44 and Fgfr2. KHSRP belongs to a ribonucleoprotein complex that includes hnRNPA1, and the two proteins cooperate in promoting epithelial-type exon usage of select pre-mRNAs. Thus, TGF-β-induced KHSRP silencing is central in a pathway leading to gene-expression changes that contribute to the cellular changes linked to EMT. PMID:27396342

  20. ChiNet uncovers rewired rewired transcription subnetworks in tolerant yeast for advanced biofuels conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conventional differential gene expression analysis is insufficient to dissect altered gene interactions for adapted transcription regulatory networks that impact downstream molecular responses. Here we present comparative chi-square network analysis (ChiNet), a computational method, to uncover rewir...

  1. Rewiring macrophages for anti-tumour immunity.

    PubMed

    Lee, Yunqin; Biswas, Subhra K

    2016-06-28

    Tumour-associated macrophages facilitate cancer progression, but whether they can be reprogrammed to elicit an anti-tumour response remains unclear. Deletion of the microRNA-processing enzyme Dicer is now shown to rewire macrophages to an anti-tumour mode, leading to an enhanced response to immunotherapy and inhibition of tumour progression. PMID:27350442

  2. The propagation of perturbations in rewired bacterial gene networks

    PubMed Central

    Baumstark, Rebecca; Hänzelmann, Sonja; Tsuru, Saburo; Schaerli, Yolanda; Francesconi, Mirko; Mancuso, Francesco M.; Castelo, Robert; Isalan, Mark

    2015-01-01

    What happens to gene expression when you add new links to a gene regulatory network? To answer this question, we profile 85 network rewirings in E. coli. Here we report that concerted patterns of differential expression propagate from reconnected hub genes. The rewirings link promoter regions to different transcription factor and σ-factor genes, resulting in perturbations that span four orders of magnitude, changing up to ∼70% of the transcriptome. Importantly, factor connectivity and promoter activity both associate with perturbation size. Perturbations from related rewirings have more similar transcription profiles and a statistical analysis reveals ∼20 underlying states of the system, associating particular gene groups with rewiring constructs. We examine two large clusters (ribosomal and flagellar genes) in detail. These represent alternative global outcomes from different rewirings because of antagonism between these major cell states. This data set of systematically related perturbations enables reverse engineering and discovery of underlying network interactions. PMID:26670742

  3. Exact solutions for network rewiring models

    NASA Astrophysics Data System (ADS)

    Evans, T. S.

    2007-03-01

    Evolving networks with a constant number of edges may be modelled using a rewiring process. These models are used to describe many real-world processes including the evolution of cultural artifacts such as family names, the evolution of gene variations, and the popularity of strategies in simple econophysics models such as the minority game. The model is closely related to Urn models used for glasses, quantum gravity and wealth distributions. The full mean field equation for the degree distribution is found and its exact solution and generating solution are given.

  4. Bacterial Virulence Proteins as Tools to Rewire Kinase Pathways in Yeast and Immune Cells

    PubMed Central

    Wei, Ping; Wong, Wilson W.; Park, Jason S.; Corcoran, Ethan E.; Peisajovich, Sergio G.; Onuffer, James J.; Weiss, Arthur; Lim, Wendell A.

    2012-01-01

    Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signaling pathways, provide a mechanism to evade immune responses during infection1,2. Although these effectors are responsible for pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behavior. Here, we have exploited two effector proteins, the Shigella flexneri OspF protein3 and Yersinia pestis YopH protein4, to systematically rewire kinase-mediated responses in both yeast and mammalian immune cells. Bacterial effector proteins can be directed to selectively inhibit specific mitogen activated protein kinase (MAPK) pathways in yeast by artificially targeting them to pathway specific complexes. Moreover, we show that unique properties of the effectors generate novel pathway behaviors: OspF, which irreversibly inactivates MAPKs4, was used to construct a synthetic feedback circuit that displays novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to precisely tune the T cell response amplitude, or as an inducible pause switch that can temporarily disable T cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications. PMID:22820255

  5. Guilt by rewiring: gene prioritization through network rewiring in Genome Wide Association Studies

    PubMed Central

    Hou, Lin; Chen, Min; Zhang, Clarence K.; Cho, Judy; Zhao, Hongyu

    2014-01-01

    Although Genome Wide Association Studies (GWAS) have identified many susceptibility loci for common diseases, they only explain a small portion of heritability. It is challenging to identify the remaining disease loci because their association signals are likely weak and difficult to identify among millions of candidates. One potentially useful direction to increase statistical power is to incorporate functional genomics information, especially gene expression networks, to prioritize GWAS signals. Most current methods utilizing network information to prioritize disease genes are based on the ‘guilt by association’ principle, in which networks are treated as static, and disease-associated genes are assumed to locate closer with each other than random pairs in the network. In contrast, we propose a novel ‘guilt by rewiring’ principle. Studying the dynamics of gene networks between controls and patients, this principle assumes that disease genes more likely undergo rewiring in patients, whereas most of the network remains unaffected in disease condition. To demonstrate this principle, we consider the changes of co-expression networks in Crohn's disease patients and controls, and how network dynamics reveals information on disease associations. Our results demonstrate that network rewiring is abundant in the immune system, and disease-associated genes are more likely to be rewired in patients. To integrate this network rewiring feature and GWAS signals, we propose to use the Markov random field framework to integrate network information to prioritize genes. Applications in Crohn's disease and Parkinson's disease show that this framework leads to more replicable results, and implicates potentially disease-associated pathways. PMID:24381306

  6. Network Evolution: Rewiring and Signatures of Conservation in Signaling

    PubMed Central

    Costanzo, Michael; Boone, Charles; Kim, Philip M.

    2012-01-01

    The analysis of network evolution has been hampered by limited availability of protein interaction data for different organisms. In this study, we investigate evolutionary mechanisms in Src Homology 3 (SH3) domain and kinase interaction networks using high-resolution specificity profiles. We constructed and examined networks for 23 fungal species ranging from Saccharomyces cerevisiae to Schizosaccharomyces pombe. We quantify rates of different rewiring mechanisms and show that interaction change through binding site evolution is faster than through gene gain or loss. We found that SH3 interactions evolve swiftly, at rates similar to those found in phosphoregulation evolution. Importantly, we show that interaction changes are sufficiently rapid to exhibit saturation phenomena at the observed timescales. Finally, focusing on the SH3 interaction network, we observe extensive clustering of binding sites on target proteins by SH3 domains and a strong correlation between the number of domains that bind a target protein (target in-degree) and interaction conservation. The relationship between in-degree and interaction conservation is driven by two different effects, namely the number of clusters that correspond to interaction interfaces and the number of domains that bind to each cluster leads to sequence specific conservation, which in turn results in interaction conservation. In summary, we uncover several network evolution mechanisms likely to generalize across peptide recognition modules. PMID:22438796

  7. Lung Adenocarcinoma Distally Rewires Hepatic Circadian Homeostasis.

    PubMed

    Masri, Selma; Papagiannakopoulos, Thales; Kinouchi, Kenichiro; Liu, Yu; Cervantes, Marlene; Baldi, Pierre; Jacks, Tyler; Sassone-Corsi, Paolo

    2016-05-01

    The circadian clock controls metabolic and physiological processes through finely tuned molecular mechanisms. The clock is remarkably plastic and adapts to exogenous "zeitgebers," such as light and nutrition. How a pathological condition in a given tissue influences systemic circadian homeostasis in other tissues remains an unanswered question of conceptual and biomedical importance. Here, we show that lung adenocarcinoma operates as an endogenous reorganizer of circadian metabolism. High-throughput transcriptomics and metabolomics revealed unique signatures of transcripts and metabolites cycling exclusively in livers of tumor-bearing mice. Remarkably, lung cancer has no effect on the core clock but rather reprograms hepatic metabolism through altered pro-inflammatory response via the STAT3-Socs3 pathway. This results in disruption of AKT, AMPK, and SREBP signaling, leading to altered insulin, glucose, and lipid metabolism. Thus, lung adenocarcinoma functions as a potent endogenous circadian organizer (ECO), which rewires the pathophysiological dimension of a distal tissue such as the liver. PAPERCLIP. PMID:27153497

  8. Adaptive rewiring aggravates the effects of species loss in ecosystems.

    PubMed

    Gilljam, David; Curtsdotter, Alva; Ebenman, Bo

    2015-01-01

    Loss of one species in an ecosystem can trigger extinctions of other dependent species. For instance, specialist predators will go extinct following the loss of their only prey unless they can change their diet. It has therefore been suggested that an ability of consumers to rewire to novel prey should mitigate the consequences of species loss by reducing the risk of cascading extinction. Using a new modelling approach on natural and computer-generated food webs we find that, on the contrary, rewiring often aggravates the effects of species loss. This is because rewiring can lead to overexploitation of resources, which eventually causes extinction cascades. Such a scenario is particularly likely if prey species cannot escape predation when rare and if predators are efficient in exploiting novel prey. Indeed, rewiring is a two-edged sword; it might be advantageous for individual predators in the short term, yet harmful for long-term system persistence. PMID:26400367

  9. Rewired Metabolism in Drug-resistant Leukemia Cells

    PubMed Central

    Stäubert, Claudia; Bhuiyan, Hasanuzzaman; Lindahl, Anna; Broom, Oliver Jay; Zhu, Yafeng; Islam, Saiful; Linnarsson, Sten; Lehtiö, Janne; Nordström, Anders

    2015-01-01

    Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies. PMID:25697355

  10. Discerning mechanistically rewired biological pathways by cumulative interaction heterogeneity statistics

    PubMed Central

    Cotton, Travis B.; Nguyen, Hien H.; Said, Joseph I.; Ouyang, Zhengyu; Zhang, Jinfa; Song, Mingzhou

    2015-01-01

    Changes in response of a biological pathway could be a consequence of either pathway rewiring, changed input, or a combination of both. Most pathway analysis methods are not designed for mechanistic rewiring such as regulatory element variations. This limits our understanding of biological pathway evolution. Here we present a Q-method to discern whether changed pathway response is caused by mechanistic rewiring of pathways due to evolution. The main innovation is a cumulative pathway interaction heterogeneity statistic accounting for rewiring-specific effects on the rate of change of each molecular variable across conditions. The Q-method remarkably outperformed differential-correlation based approaches on data from diverse biological processes. Strikingly, it also worked well in differentiating rewired chaotic systems, whose dynamics are notoriously difficult to predict. Applying the Q-method on transcriptome data of four yeasts, we show that pathway interaction heterogeneity for known metabolic and signaling pathways is indeed a predictor of interspecies genetic rewiring due to unbalanced TATA box-containing genes among the yeasts. The demonstrated effectiveness of the Q-method paves the way to understanding network evolution at the resolution of functional biological pathways. PMID:25921728

  11. Discerning mechanistically rewired biological pathways by cumulative interaction heterogeneity statistics.

    PubMed

    Cotton, Travis B; Nguyen, Hien H; Said, Joseph I; Ouyang, Zhengyu; Zhang, Jinfa; Song, Mingzhou

    2015-01-01

    Changes in response of a biological pathway could be a consequence of either pathway rewiring, changed input, or a combination of both. Most pathway analysis methods are not designed for mechanistic rewiring such as regulatory element variations. This limits our understanding of biological pathway evolution. Here we present a Q-method to discern whether changed pathway response is caused by mechanistic rewiring of pathways due to evolution. The main innovation is a cumulative pathway interaction heterogeneity statistic accounting for rewiring-specific effects on the rate of change of each molecular variable across conditions. The Q-method remarkably outperformed differential-correlation based approaches on data from diverse biological processes. Strikingly, it also worked well in differentiating rewired chaotic systems, whose dynamics are notoriously difficult to predict. Applying the Q-method on transcriptome data of four yeasts, we show that pathway interaction heterogeneity for known metabolic and signaling pathways is indeed a predictor of interspecies genetic rewiring due to unbalanced TATA box-containing genes among the yeasts. The demonstrated effectiveness of the Q-method paves the way to understanding network evolution at the resolution of functional biological pathways. PMID:25921728

  12. Rewiring of Cellular Membrane Homeostasis by Picornaviruses

    PubMed Central

    2014-01-01

    Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway “as is” for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. PMID:24920802

  13. Phosphorylation network rewiring by gene duplication

    PubMed Central

    Freschi, Luca; Courcelles, Mathieu; Thibault, Pierre; Michnick, Stephen W; Landry, Christian R

    2011-01-01

    Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post-translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post-duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase–substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post-translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them. PMID:21734643

  14. Phase transitions in a coevolving snowdrift game with costly rewiring

    NASA Astrophysics Data System (ADS)

    Zhang, W.; Li, Y. S.; Du, P.; Xu, C.; Hui, P. M.

    2014-11-01

    We propose and study a dissatisfied adaptive snowdrift game with a payoff parameter r that incorporates a cost for rewiring a connection. An agent, facing adverse local environment, may switch action without a cost or rewire an existing link with a cost a so as to attain a better competing environment. Detailed numerical simulations reveal nontrivial and nonmonotonic dependence of the frequency of cooperation and the densities of different types of links on a and r . A theory that treats the cooperative and noncooperative agents separately and accounts for spatial correlation up to neighboring agents is formulated. The theory gives results that are in good agreement with simulations. The frequency of cooperation fC is enhanced (suppressed) at high rewiring cost relative to that at low rewiring cost when r is small (large). For a given value of r , there exists a critical value of the rewiring cost below which the system evolves into a phase of frozen dynamics with isolated noncooperative agents segregated from a cluster of cooperative agents, and above which the system evolves into a connected population of mixed actions with continual dynamics. The phase boundary on the a -r phase space that separates the two phases with distinct structural, population and dynamical properties is mapped out. The phase diagram reveals that, as a general feature, for small r (small a ), the disconnected and segregated phase can survive over a wider range of a (r ) .

  15. Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming.

    PubMed

    Beagan, Jonathan A; Gilgenast, Thomas G; Kim, Jesi; Plona, Zachary; Norton, Heidi K; Hu, Gui; Hsu, Sarah C; Shields, Emily J; Lyu, Xiaowen; Apostolou, Effie; Hochedlinger, Konrad; Corces, Victor G; Dekker, Job; Phillips-Cremins, Jennifer E

    2016-05-01

    Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression. PMID:27152443

  16. Rewiring Neural Interactions by Micro-Stimulation

    PubMed Central

    Rebesco, James M.; Stevenson, Ian H.; Körding, Konrad P.; Solla, Sara A.; Miller, Lee E.

    2010-01-01

    Plasticity is a crucial component of normal brain function and a critical mechanism for recovery from injury. In vitro, associative pairing of presynaptic spiking and stimulus-induced postsynaptic depolarization causes changes in the synaptic efficacy of the presynaptic neuron, when activated by extrinsic stimulation. In vivo, such paradigms can alter the responses of whole groups of neurons to stimulation. Here, we used in vivo spike-triggered stimulation to drive plastic changes in rat forelimb sensorimotor cortex, which we monitored using a statistical measure of functional connectivity inferred from the spiking statistics of the neurons during normal, spontaneous behavior. These induced plastic changes in inferred functional connectivity depended on the latency between trigger spike and stimulation, and appear to reflect a robust reorganization of the network. Such targeted connectivity changes might provide a tool for rerouting the flow of information through a network, with implications for both rehabilitation and brain–machine interface applications. PMID:20838477

  17. A Nanoparticle-Based Combination Chemotherapy Delivery System for Enhanced Tumor Killing by Dynamic Rewiring of Signaling Pathways

    PubMed Central

    Morton, Stephen W.; Lee, Michael J.; Deng, Zhou J.; Dreaden, Erik C.; Siouve, Elise; Shopsowitz, Kevin E.; Shah, Nisarg J.; Yaffe, Michael B.; Hammond, Paula T.

    2014-01-01

    Exposure to the EGFR (epidermal growth factor receptor) inhibitor erlotinib promotes the dynamic rewiring of apoptotic pathways, which sensitizes cells within a specific period to subsequent exposure to the DNA-damaging agent doxorubicin. A critical challenge for translating this therapeutic network rewiring into clinical practice is the design of optimal drug delivery systems. We report the generation of a nanoparticle delivery vehicle that contained more than one therapeutic agent and produced a controlled sequence of drug release. Liposomes, representing the first clinically approved nanomedicine systems, are well-characterized, simple, and versatile platforms for the manufacture of functional and tunable drug carriers. Using the hydrophobic and hydrophilic compartments of liposomes, we effectively incorporated both hydrophobic (erlotinib) and hydrophilic (doxorubicin) small molecules, through which we achieved the desired time sequence of drug release. We also coated the liposomes with folate to facilitate targeting to cancer cells. When compared to the time-staggered application of individual drugs, staggered release from tumor-targeted single liposomal particles enhanced dynamic rewiring of apoptotic signaling pathways, resulting in improved tumor cell killing in culture and tumor shrinkage in animal models. PMID:24825919

  18. Linalool is a PPARα ligand that reduces plasma TG levels and rewires the hepatic transcriptome and plasma metabolome[S

    PubMed Central

    Jun, Hee-jin; Lee, Ji Hae; Kim, Jiyoung; Jia, Yaoyao; Kim, Kyoung Heon; Hwang, Kwang Yeon; Yun, Eun Ju; Do, Kyoung-Rok; Lee, Sung-Joon

    2014-01-01

    We investigated the hypotriglyceridemic mechanism of action of linalool, an aromatic monoterpene present in teas and fragrant herbs. Reporter gene and time-resolved fluorescence resonance energy transfer assays demonstrated that linalool is a direct ligand of PPARα. Linalool stimulation reduced cellular lipid accumulation regulating PPARα-responsive genes and significantly induced FA oxidation, and its effects were markedly attenuated by silencing PPARα expression. In mice, the oral administration of linalool for 3 weeks reduced plasma TG concentrations in Western-diet-fed C57BL/6J mice (31%, P < 0.05) and human apo E2 mice (50%, P < 0.05) and regulated hepatic PPARα target genes. However, no such effects were seen in PPARα-deficient mice. Transcriptome profiling revealed that linalool stimulation rewired global gene expression in lipid-loaded hepatocytes and that the effects of 1 mM linalool were comparable to those of 0.1 mM fenofibrate. Metabolomic analysis of the mouse plasma revealed that the global metabolite profiles were significantly distinguishable between linalool-fed mice and controls. Notably, the concentrations of saturated FAs were significantly reduced in linalool-fed mice. These findings suggest that the appropriate intake of a natural aromatic compound could exert beneficial metabolic effects by regulating a cellular nutrient sensor. PMID:24752549

  19. Linalool is a PPARα ligand that reduces plasma TG levels and rewires the hepatic transcriptome and plasma metabolome.

    PubMed

    Jun, Hee-Jin; Lee, Ji Hae; Kim, Jiyoung; Jia, Yaoyao; Kim, Kyoung Heon; Hwang, Kwang Yeon; Yun, Eun Ju; Do, Kyoung-Rok; Lee, Sung-Joon

    2014-06-01

    We investigated the hypotriglyceridemic mechanism of action of linalool, an aromatic monoterpene present in teas and fragrant herbs. Reporter gene and time-resolved fluorescence resonance energy transfer assays demonstrated that linalool is a direct ligand of PPARα. Linalool stimulation reduced cellular lipid accumulation regulating PPARα-responsive genes and significantly induced FA oxidation, and its effects were markedly attenuated by silencing PPARα expression. In mice, the oral administration of linalool for 3 weeks reduced plasma TG concentrations in Western-diet-fed C57BL/6J mice (31%, P < 0.05) and human apo E2 mice (50%, P < 0.05) and regulated hepatic PPARα target genes. However, no such effects were seen in PPARα-deficient mice. Transcriptome profiling revealed that linalool stimulation rewired global gene expression in lipid-loaded hepatocytes and that the effects of 1 mM linalool were comparable to those of 0.1 mM fenofibrate. Metabolomic analysis of the mouse plasma revealed that the global metabolite profiles were significantly distinguishable between linalool-fed mice and controls. Notably, the concentrations of saturated FAs were significantly reduced in linalool-fed mice. These findings suggest that the appropriate intake of a natural aromatic compound could exert beneficial metabolic effects by regulating a cellular nutrient sensor. PMID:24752549

  20. Topological, functional, and dynamic properties of the protein interaction networks rewired by benzo(a)pyrene

    SciTech Connect

    Ba, Qian; Li, Junyang; Huang, Chao; Li, Jingquan; Chu, Ruiai; Wu, Yongning; Wang, Hui

    2015-03-01

    Benzo(a)pyrene is a common environmental and foodborne pollutant that has been identified as a human carcinogen. Although the carcinogenicity of benzo(a)pyrene has been extensively reported, its precise molecular mechanisms and the influence on system-level protein networks are not well understood. To investigate the system-level influence of benzo(a)pyrene on protein interactions and regulatory networks, a benzo(a)pyrene-rewired protein interaction network was constructed based on 769 key proteins derived from more than 500 literature reports. The protein interaction network rewired by benzo(a)pyrene was a scale-free, highly-connected biological system. Ten modules were identified, and 25 signaling pathways were enriched, most of which belong to the human diseases category, especially cancer and infectious disease. In addition, two lung-specific and two liver-specific pathways were identified. Three pathways were specific in short and medium-term networks (< 48 h), and five pathways were enriched only in the medium-term network (6 h–48 h). Finally, the expression of linker genes in the network was validated by Western blotting. These findings establish the overall, tissue- and time-specific benzo(a)pyrene-rewired protein interaction networks and provide insights into the biological effects and molecular mechanisms of action of benzo(a)pyrene. - Highlights: • Benzo(a)pyrene induced scale-free, highly-connected protein interaction networks. • 25 signaling pathways were enriched through modular analysis. • Tissue- and time-specific pathways were identified.

  1. ChiNet uncovers rewired transcription subnetworks in tolerant yeast for advanced biofuels conversion

    PubMed Central

    Zhang, Yang; Liu, Z. Lewis; Song, Mingzhou

    2015-01-01

    Analysis of rewired upstream subnetworks impacting downstream differential gene expression aids the delineation of evolving molecular mechanisms. Cumulative statistics based on conventional differential correlation are limited for subnetwork rewiring analysis since rewiring is not necessarily equivalent to change in correlation coefficients. Here we present a computational method ChiNet to quantify subnetwork rewiring by statistical heterogeneity that enables detection of potential genotype changes causing altered transcription regulation in evolving organisms. Given a differentially expressed downstream gene set, ChiNet backtracks a rewired upstream subnetwork from a super-network including gene interactions known to occur under various molecular contexts. We benchmarked ChiNet for its high accuracy in distinguishing rewired artificial subnetworks, in silico yeast transcription-metabolic subnetworks, and rewired transcription subnetworks for Candida albicans versus Saccharomyces cerevisiae, against two differential-correlation based subnetwork rewiring approaches. Then, using transcriptome data from tolerant S. cerevisiae strain NRRL Y-50049 and a wild-type intolerant strain, ChiNet identified 44 metabolic pathways affected by rewired transcription subnetworks anchored to major adaptively activated transcription factor genes YAP1, RPN4, SFP1 and ROX1, in response to toxic chemical challenges involved in lignocellulose-to-biofuels conversion. These findings support the use of ChiNet in rewiring analysis of subnetworks where differential interaction patterns resulting from divergent nonlinear dynamics abound. PMID:25897127

  2. Targeted gene silencing to induce permanent sterility.

    PubMed

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

    2012-08-01

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

  3. Effects of random rewiring on the degree correlation of scale-free networks

    NASA Astrophysics Data System (ADS)

    Qu, Jing; Wang, Sheng-Jun; Jusup, Marko; Wang, Zhen

    2015-10-01

    Random rewiring is used to generate null networks for the purpose of analyzing the topological properties of scale-free networks, yet the effects of random rewiring on the degree correlation are subject to contradicting interpretations in the literature. We comprehensively analyze the degree correlation of randomly rewired scale-free networks and show that random rewiring increases disassortativity by reducing the average degree of the nearest neighbors of high-degree nodes. The effect can be captured by the measures of the degree correlation that consider all links in the network, but not by analogous measures that consider only links between degree peers, hence the potential for contradicting interpretations. We furthermore find that random and directional rewiring affect the topology of a scale-free network differently, even if the degree correlation of the rewired networks is the same. Consequently, the network dynamics is changed, which is proven here by means of the biased random walk.

  4. Effects of random rewiring on the degree correlation of scale-free networks

    PubMed Central

    Qu, Jing; Wang, Sheng-Jun; Jusup, Marko; Wang, Zhen

    2015-01-01

    Random rewiring is used to generate null networks for the purpose of analyzing the topological properties of scale-free networks, yet the effects of random rewiring on the degree correlation are subject to contradicting interpretations in the literature. We comprehensively analyze the degree correlation of randomly rewired scale-free networks and show that random rewiring increases disassortativity by reducing the average degree of the nearest neighbors of high-degree nodes. The effect can be captured by the measures of the degree correlation that consider all links in the network, but not by analogous measures that consider only links between degree peers, hence the potential for contradicting interpretations. We furthermore find that random and directional rewiring affect the topology of a scale-free network differently, even if the degree correlation of the rewired networks is the same. Consequently, the network dynamics is changed, which is proven here by means of the biased random walk. PMID:26482005

  5. Exact solution for the time evolution of network rewiring models

    NASA Astrophysics Data System (ADS)

    Evans, T. S.; Plato, A. D. K.

    2007-05-01

    We consider the rewiring of a bipartite graph using a mixture of random and preferential attachment. The full mean-field equations for the degree distribution and its generating function are given. The exact solution of these equations for all finite parameter values at any time is found in terms of standard functions. It is demonstrated that these solutions are an excellent fit to numerical simulations of the model. We discuss the relationship between our model and several others in the literature, including examples of urn, backgammon, and balls-in-boxes models, the Watts and Strogatz rewiring problem, and some models of zero range processes. Our model is also equivalent to those used in various applications including cultural transmission, family name and gene frequencies, glasses, and wealth distributions. Finally some Voter models and an example of a minority game also show features described by our model.

  6. Re-engineering cellular physiology by rewiring high-level global regulatory genes

    PubMed Central

    Fitzgerald, Stephen; Dillon, Shane C.; Chao, Tzu-Chiao; Wiencko, Heather L.; Hokamp, Karsten; Cameron, Andrew D. S.; Dorman, Charles J.

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  7. Re-engineering cellular physiology by rewiring high-level global regulatory genes.

    PubMed

    Fitzgerald, Stephen; Dillon, Shane C; Chao, Tzu-Chiao; Wiencko, Heather L; Hokamp, Karsten; Cameron, Andrew D S; Dorman, Charles J

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  8. Axin cancer mutants form nanoaggregates to rewire the Wnt signaling network.

    PubMed

    Anvarian, Zeinab; Nojima, Hisashi; van Kappel, Eline C; Madl, Tobias; Spit, Maureen; Viertler, Martin; Jordens, Ingrid; Low, Teck Y; van Scherpenzeel, Revina C; Kuper, Ineke; Richter, Klaus; Heck, Albert J R; Boelens, Rolf; Vincent, Jean-Paul; Rüdiger, Stefan G D; Maurice, Madelon M

    2016-04-01

    Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment. PMID:26974125

  9. Gene Network Rewiring to Study Melanoma Stage Progression and Elements Essential for Driving Melanoma

    PubMed Central

    Kaushik, Abhinav; Bhatia, Yashuma; Ali, Shakir; Gupta, Dinesh

    2015-01-01

    Metastatic melanoma patients have a poor prognosis, mainly attributable to the underlying heterogeneity in melanoma driver genes and altered gene expression profiles. These characteristics of melanoma also make the development of drugs and identification of novel drug targets for metastatic melanoma a daunting task. Systems biology offers an alternative approach to re-explore the genes or gene sets that display dysregulated behaviour without being differentially expressed. In this study, we have performed systems biology studies to enhance our knowledge about the conserved property of disease genes or gene sets among mutually exclusive datasets representing melanoma progression. We meta-analysed 642 microarray samples to generate melanoma reconstructed networks representing four different stages of melanoma progression to extract genes with altered molecular circuitry wiring as compared to a normal cellular state. Intriguingly, a majority of the melanoma network-rewired genes are not differentially expressed and the disease genes involved in melanoma progression consistently modulate its activity by rewiring network connections. We found that the shortlisted disease genes in the study show strong and abnormal network connectivity, which enhances with the disease progression. Moreover, the deviated network properties of the disease gene sets allow ranking/prioritization of different enriched, dysregulated and conserved pathway terms in metastatic melanoma, in agreement with previous findings. Our analysis also reveals presence of distinct network hubs in different stages of metastasizing tumor for the same set of pathways in the statistically conserved gene sets. The study results are also presented as a freely available database at http://bioinfo.icgeb.res.in/m3db/. The web-based database resource consists of results from the analysis presented here, integrated with cytoscape web and user-friendly tools for visualization, retrieval and further analysis. PMID

  10. Rewiring the RNAs of influenza virus to prevent reassortment

    PubMed Central

    Gao, Qinshan; Palese, Peter

    2009-01-01

    Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses. PMID:19805230

  11. Polarized nuclear target based on parahydrogen induced polarization

    SciTech Connect

    D. Budker, M.P. Ledbetter, S. Appelt, L.S. Bouchard, B. Wojtsekhowski

    2012-12-01

    We discuss a novel concept of a polarized nuclear target for accelerator fixed-target scattering experiments, which is based on parahydrogen induced polarization (PHIP). One may be able to reach a 33% free-proton polarization in the ethane molecule. The potential advantages of such a target include operation at zero magnetic field, fast ({approx}100 HZ) polarization oscillation (akin to polarization reversal), and operation with large intensity of an electron beam.

  12. Polarized nuclear target based on parahydrogen induced polarization

    NASA Astrophysics Data System (ADS)

    Budker, D.; Ledbetter, M. P.; Appelt, S.; Bouchard, L. S.; Wojtsekhowski, B.

    2012-12-01

    We discuss a novel concept of a polarized nuclear target for accelerator fixed-target scattering experiments, which is based on parahydrogen induced polarization (PHIP). One may be able to reach a 33% free-proton polarization in the ethane molecule. The potential advantages of such a target include operation at zero magnetic field, fast (˜100 Hz) polarization oscillation (akin to polarization reversal), and operation with large intensity of an electron beam.

  13. Micro-rewiring as a substrate for learning.

    PubMed

    DeBello, William M

    2008-11-01

    How does the brain encode life experiences? Recent results derived from vital imaging, computational modeling, cellular physiology and systems neuroscience have pointed to local changes in synaptic connectivity as a powerful substrate, here termed micro-rewiring. To examine this hypothesis, I first review findings on micro-structural dynamics with focus on the extension and retraction of dendritic spines. Although these observations demonstrate a biological mechanism, they do not inform us of the specific changes in circuit configuration that might occur during learning. Here, computational models have made testable predictions for both the neuronal and circuit levels. Integrative approaches in the mammalian neocortex and the barn owl auditory localization pathway provide some of the first direct evidence in support of these 'synaptic-clustering' mechanisms. The implications of these data and the challenges for future research are discussed. PMID:18817991

  14. Inverse relationship between adult hippocampal cell proliferation and synaptic rewiring in the dentate gyrus.

    PubMed

    Butz, Markus; Teuchert-Noodt, Gertraud; Grafen, Keren; van Ooyen, Arjen

    2008-01-01

    Adult neurogenesis is a key feature of the hippocampal dentate gyrus (DG). Neurogenesis is accompanied by synaptogenesis as new cells become integrated into the circuitry of the hippocampus. However, little is known to what extent the embedding of new neurons rewires the pre-existing network. Here we investigate synaptic rewiring in the DG of gerbils (Meriones unguiculatus) under different rates of adult cell proliferation caused by different rearing conditions as well as juvenile methamphetamine treatment. Surprisingly, we found that an increased cell proliferation reduced the amount of synaptic rewiring. To help explain this unexpected finding, we developed a novel model of dentate network formation incorporating neurogenesis and activity-dependent synapse formation and remodelling. In the model, we show that homeostasis of neuronal activity can account for the inverse relationship between cell proliferation and synaptic rewiring. PMID:18481284

  15. Kinome-wide decoding of network-attacking mutations rewiring cancer signaling.

    PubMed

    Creixell, Pau; Schoof, Erwin M; Simpson, Craig D; Longden, James; Miller, Chad J; Lou, Hua Jane; Perryman, Lara; Cox, Thomas R; Zivanovic, Nevena; Palmeri, Antonio; Wesolowska-Andersen, Agata; Helmer-Citterich, Manuela; Ferkinghoff-Borg, Jesper; Itamochi, Hiroaki; Bodenmiller, Bernd; Erler, Janine T; Turk, Benjamin E; Linding, Rune

    2015-09-24

    Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks. PMID:26388441

  16. Kinome-wide Decoding of Network-Attacking Mutations Rewiring Cancer Signaling

    PubMed Central

    Creixell, Pau; Schoof, Erwin M.; Simpson, Craig D.; Longden, James; Miller, Chad J.; Lou, Hua Jane; Perryman, Lara; Cox, Thomas R.; Zivanovic, Nevena; Palmeri, Antonio; Wesolowska-Andersen, Agata; Helmer-Citterich, Manuela; Ferkinghoff-Borg, Jesper; Itamochi, Hiroaki; Bodenmiller, Bernd; Erler, Janine T.; Turk, Benjamin E.; Linding, Rune

    2015-01-01

    Summary Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks. PMID:26388441

  17. Regulatory network rewiring for secondary metabolism in Arabidopsis thaliana under various conditions

    PubMed Central

    2014-01-01

    Background Plant secondary metabolites are critical to various biological processes. However, the regulations of these metabolites are complex because of regulatory rewiring or crosstalk. To unveil how regulatory behaviors on secondary metabolism reshape biological processes, we constructed and analyzed a dynamic regulatory network of secondary metabolic pathways in Arabidopsis. Results The dynamic regulatory network was constructed through integrating co-expressed gene pairs and regulatory interactions. Regulatory interactions were either predicted by conserved transcription factor binding sites (TFBSs) or proved by experiments. We found that integrating two data (co-expression and predicted regulatory interactions) enhanced the number of highly confident regulatory interactions by over 10% compared with using single data. The dynamic changes of regulatory network systematically manifested regulatory rewiring to explain the mechanism of regulation, such as in terpenoids metabolism, the regulatory crosstalk of RAV1 (AT1G13260) and ATHB1 (AT3G01470) on HMG1 (hydroxymethylglutaryl-CoA reductase, AT1G76490); and regulation of RAV1 on epoxysqualene biosynthesis and sterol biosynthesis. Besides, we investigated regulatory rewiring with expression, network topology and upstream signaling pathways. Regulatory rewiring was revealed by the variability of genes’ expression: pathway genes and transcription factors (TFs) were significantly differentially expressed under different conditions (such as terpenoids biosynthetic genes in tissue experiments and E2F/DP family members in genotype experiments). Both network topology and signaling pathways supported regulatory rewiring. For example, we discovered correlation among the numbers of pathway genes, TFs and network topology: one-gene pathways (such as δ-carotene biosynthesis) were regulated by a fewer TFs, and were not critical to metabolic network because of their low degrees in topology. Upstream signaling pathways of 50

  18. Mechanism of genotoxicity induced by targeted cytoplasmic irradiation

    PubMed Central

    Hong, M; Xu, A; Zhou, H; Wu, L; Randers-Pehrson, G; Santella, R M; Yu, Z; Hei, T K

    2010-01-01

    Background: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown. Methods: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human–hamster hybrid (AL) cells. Results: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in AL cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of AL cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway. Conclusion: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect. PMID:20842121

  19. Targeting Glutamine Induces Apoptosis: A Cancer Therapy Approach

    PubMed Central

    Chen, Lian; Cui, Hengmin

    2015-01-01

    Glutamine metabolism has been proved to be dysregulated in many cancer cells, and is essential for proliferation of most cancer cells, which makes glutamine an appealing target for cancer therapy. In order to be well used by cells, glutamine must be transported to cells by specific transporters and converted to glutamate by glutaminase. There are currently several drugs that target glutaminase under development or clinical trials. Also, glutamine metabolism restriction has been proved to be effective in inhibiting tumor growth both in vivo and vitro through inducing apoptosis, growth arrest and/or autophagy. Here, we review recent researches about glutamine metabolism in cancer, and cell death induced by targeting glutamine, and their potential roles in cancer therapy. PMID:26402672

  20. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  1. Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling

    PubMed Central

    Podgornaia, Anna I.; Casino, Patricia; Marina, Alberto; Laub, Michael T.

    2013-01-01

    Summary Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, E. coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes, along with a systematic mutagenesis study, reveals how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions, protein evolution, and the design of novel protein interfaces. PMID:23954504

  2. Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling.

    PubMed

    Podgornaia, Anna I; Casino, Patricia; Marina, Alberto; Laub, Michael T

    2013-09-01

    Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces. PMID:23954504

  3. Naringenin targets ERK2 and suppresses UVB-induced photoaging.

    PubMed

    Jung, Sung Keun; Ha, Su Jeong; Jung, Chang Hwa; Kim, Yun Tai; Lee, Hoo-Keun; Kim, Myoung Ok; Lee, Mee-Hyun; Mottamal, Madhusoodanan; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2016-05-01

    A number of natural phytochemicals have anti-photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase-1 (MMP-1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB-induced MMP-1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3-dimensional (3-D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm(2) ) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB-induced MMP-1 expression and AP-1 activity, and strongly suppressed UVB-induced phosphorylation of Fos-related antigen (FRA)-1 at Ser265. Importantly, UVB irradiation-induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen-activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB-induced extracellular signal-regulated kinase 2 (ERK2) activity and subsequently attenuated UVB-induced phosphorylation of p90(RSK) by competitively binding with ATP. Constitutively active MEK (CA-MEK) increased FRA1 phosphorylation and expression and also induced MMP-1 expression, whereas dominant-negative ERK2 (DN-ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP-1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB-induced wrinkle formation, trans-epidermal water loss and MMP-13 expression. Naringenin exerts potent anti-photoaging effects by suppressing ERK2

  4. Piperlongumine induces autophagy by targeting p38 signaling.

    PubMed

    Wang, Y; Wang, J-W; Xiao, X; Shan, Y; Xue, B; Jiang, G; He, Q; Chen, J; Xu, H-G; Zhao, R-X; Werle, K D; Cui, R; Liang, J; Li, Y-L; Xu, Z-X

    2013-01-01

    Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death. PMID:24091667

  5. Enhancing the synchronizability of networks by rewiring based on tabu search and a local greedy algorithm

    NASA Astrophysics Data System (ADS)

    Yang, Cui-Li; Tang, Kit-Sang

    2011-12-01

    By considering the eigenratio of the Laplacian matrix as the synchronizability measure, this paper presents an efficient method to enhance the synchronizability of undirected and unweighted networks via rewiring. The rewiring method combines the use of tabu search and a local greedy algorithm so that an effective search of solutions can be achieved. As demonstrated in the simulation results, the performance of the proposed approach outperforms the existing methods for a large variety of initial networks, both in terms of speed and quality of solutions.

  6. Rewiring of the Ppr1 Zinc Cluster Transcription Factor from Purine Catabolism to Pyrimidine Biogenesis in the Saccharomycetaceae.

    PubMed

    Tebung, Walters Aji; Choudhury, Baharul I; Tebbji, Faiza; Morschhäuser, Joachim; Whiteway, Malcolm

    2016-07-11

    Metabolic pathways are largely conserved in eukaryotes, but the transcriptional regulation of these pathways can sometimes vary between species; this has been termed "rewiring." Recently, it has been established that in the Saccharomyces lineage starting from Naumovozyma castellii, genes involved in allantoin breakdown have been genomically relocated to form the DAL cluster. The formation of the DAL cluster occurred along with the loss of urate permease (UAP) and urate oxidase (UOX), reducing the requirement for oxygen and bypassing the candidate Ppr1 inducer, uric acid. In Saccharomyces cerevisiae, this allantoin catabolism cluster is regulated by the transcription factor Dal82, which is not present in many of the pre-rearrangement fungal species. We have used ChIP-chip analysis, transcriptional profiling of an activated Ppr1 protein, bioinformatics, and nitrogen utilization studies to establish that in Candida albicans the zinc cluster transcription factor Ppr1 controls this allantoin catabolism regulon. Intriguingly, in S. cerevisiae, the Ppr1 ortholog binds the same DNA motif (CGG(N6)CCG) as in C. albicans but serves as a regulator of pyrimidine biosynthesis. This transcription factor rewiring appears to have taken place at the same phylogenetic step as the formation of the rearranged DAL cluster. This transfer of the control of allantoin degradation from Ppr1 to Dal82, together with the repositioning of Ppr1 to the regulation of pyrimidine biosynthesis, may have resulted from a switch to a metabolism that could exploit hypoxic conditions in the lineage leading to N. castellii and S. cerevisiae. PMID:27321996

  7. Non-targeted effects induced by high LET charged particles

    NASA Astrophysics Data System (ADS)

    Hei, Tom K.; Chai, Yunfei; Hamada, Nobuyuki; Kakinuma, Shizuko; Uchihori, Yukio

    Radiation-induced non-targeted response represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the final biological consequences of exposure to low doses of radiation. Using the gpt delta transgenic mouse model, there is evidence that irradiation of a small area (1 cm by 1 cm) of the lower abdominal area of animals with a 5 Gy dose of X-rays induced cyclooxygenase-2 as well as deletion mutations in the out-of-field lung tissues of the animals. The mutation correlated with an increase in prostaglandin levels in the bystander lung tissues and with an increase in the level of 8-hydroxydeoxyguanosine (8-OHdG), an oxidative DNA damage marker. An increase in COX-2 level was also detected in the out-of-field lung tissues of animals similarly exposed to high LET argon and carbon ions accelerated at the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences in Japan. These results provide the first evidence that the COX-2 -related pathway, which is essential in mediating cellular inflammatory response, is the critical signaling link for the non-targeted, bystander phenomenon. A better understanding of the cellular and molecular mechanisms of the non-targeted, out of field phenomenon together with evidence of their occurrence in vivo will allow us to formulate a more accurate assessment of radiation risk.

  8. Induced oligomerization targets Golgi proteins for degradation in lysosomes

    PubMed Central

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D.

    2015-01-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  9. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  10. β-catenin is central to DUX4-driven network rewiring in facioscapulohumeral muscular dystrophy

    PubMed Central

    Banerji, Christopher R. S.; Knopp, Paul; Moyle, Louise A.; Severini, Simone; Orrell, Richard W.; Teschendorff, Andrew E.; Zammit, Peter S.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an incurable disease, characterized by skeletal muscle weakness and wasting. Genetically, FSHD is characterized by contraction or hypomethylation of repeat D4Z4 units on chromosome 4, which causes aberrant expression of the transcription factor DUX4 from the last repeat. Many genes have been implicated in FSHD pathophysiology, but an integrated molecular model is currently lacking. We developed a novel differential network methodology, Interactome Sparsification and Rewiring (InSpiRe), which detects network rewiring between phenotypes by integrating gene expression data with known protein interactions. Using InSpiRe, we performed a meta-analysis of multiple microarray datasets from FSHD muscle biopsies, then removed secondary rewiring using non-FSHD datasets, to construct a unified network of rewired interactions. Our analysis identified β-catenin as the main coordinator of FSHD-associated protein interaction signalling, with pathways including canonical Wnt, HIF1-α and TNF-α clearly perturbed. To detect transcriptional changes directly elicited by DUX4, gene expression profiling was performed using microarrays on murine myoblasts. This revealed that DUX4 significantly modified expression of the genes in our FSHD network. Furthermore, we experimentally confirmed that Wnt/β-catenin signalling is affected by DUX4 in murine myoblasts. Thus, we provide the first unified molecular map of FSHD signalling, capable of uncovering pathomechanisms and guiding therapeutic development. PMID:25551153

  11. β-Catenin is central to DUX4-driven network rewiring in facioscapulohumeral muscular dystrophy.

    PubMed

    Banerji, Christopher R S; Knopp, Paul; Moyle, Louise A; Severini, Simone; Orrell, Richard W; Teschendorff, Andrew E; Zammit, Peter S

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an incurable disease, characterized by skeletal muscle weakness and wasting. Genetically, FSHD is characterized by contraction or hypomethylation of repeat D4Z4 units on chromosome 4, which causes aberrant expression of the transcription factor DUX4 from the last repeat. Many genes have been implicated in FSHD pathophysiology, but an integrated molecular model is currently lacking. We developed a novel differential network methodology, Interactome Sparsification and Rewiring (InSpiRe), which detects network rewiring between phenotypes by integrating gene expression data with known protein interactions. Using InSpiRe, we performed a meta-analysis of multiple microarray datasets from FSHD muscle biopsies, then removed secondary rewiring using non-FSHD datasets, to construct a unified network of rewired interactions. Our analysis identified β-catenin as the main coordinator of FSHD-associated protein interaction signalling, with pathways including canonical Wnt, HIF1-α and TNF-α clearly perturbed. To detect transcriptional changes directly elicited by DUX4, gene expression profiling was performed using microarrays on murine myoblasts. This revealed that DUX4 significantly modified expression of the genes in our FSHD network. Furthermore, we experimentally confirmed that Wnt/β-catenin signalling is affected by DUX4 in murine myoblasts. Thus, we provide the first unified molecular map of FSHD signalling, capable of uncovering pathomechanisms and guiding therapeutic development. PMID:25551153

  12. Targeted genes and interacting proteins of hypoxia inducible factor-1

    PubMed Central

    Liu, Wei; Shen, Shao-Ming; Zhao, Xu-Yun; Chen, Guo-Qiang

    2012-01-01

    Heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1) functions as a master regulator of oxygen homeostasis in almost all nucleated mammalian cells. The fundamental process adapted to cellular oxygen alteration largely depends on the refined regulation on its alpha subunit, HIF-1α. Recent studies have unraveled expanding and critical roles of HIF-1α, involving in a multitude of developmental, physiological, and pathophysiological processes. This review will focus on the current knowledge of HIF-1α-targeting genes and its interacting proteins, as well as the concomitant functional relationships between them. PMID:22773957

  13. Dynamics of the cell-cycle network under genome-rewiring perturbations

    NASA Astrophysics Data System (ADS)

    Katzir, Yair; Elhanati, Yuval; Averbukh, Inna; Braun, Erez

    2013-12-01

    The cell-cycle progression is regulated by a specific network enabling its ordered dynamics. Recent experiments supported by computational models have shown that a core of genes ensures this robust cycle dynamics. However, much less is known about the direct interaction of the cell-cycle regulators with genes outside of the cell-cycle network, in particular those of the metabolic system. Following our recent experimental work, we present here a model focusing on the dynamics of the cell-cycle core network under rewiring perturbations. Rewiring is achieved by placing an essential metabolic gene exclusively under the regulation of a cell-cycle's promoter, forcing the cell-cycle network to function under a multitasking challenging condition; operating in parallel the cell-cycle progression and a metabolic essential gene. Our model relies on simple rate equations that capture the dynamics of the relevant protein-DNA and protein-protein interactions, while making a clear distinction between these two different types of processes. In particular, we treat the cell-cycle transcription factors as limited ‘resources’ and focus on the redistribution of resources in the network during its dynamics. This elucidates the sensitivity of its various nodes to rewiring interactions. The basic model produces the correct cycle dynamics for a wide range of parameters. The simplicity of the model enables us to study the interface between the cell-cycle regulation and other cellular processes. Rewiring a promoter of the network to regulate a foreign gene, forces a multitasking regulatory load. The higher the load on the promoter, the longer is the cell-cycle period. Moreover, in agreement with our experimental results, the model shows that different nodes of the network exhibit variable susceptibilities to the rewiring perturbations. Our model suggests that the topology of the cell-cycle core network ensures its plasticity and flexible interface with other cellular processes, without

  14. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  15. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response.

    PubMed

    Harwardt, Thomas; Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C; Nevels, Michael; Paulus, Christina

    2016-07-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  16. Radioactive targets for neutron-induced cross section measurements

    SciTech Connect

    Kronenberg, A.; Bond, E. M.; Glover, S. E.; Rundberg, R. S.; Vieira, D. J.; Esch, E. I.; Reifarth, R.; Ullmann, J. L.; Haight, Robert C.; Rochmann, D.

    2004-01-01

    Measurements using radioactive targets are important for the determination of key reaction path ways associated with the synthesis of the elements in nuclear astrophysics (sprocess), advanced fuel cycle initiative (transmutation of radioactive waste), and stockpile stewardship. High precision capture cross-section measurements are needed to interpret observations, predict elemental or isotopical ratios, and unobserved abundances. There are two new detector systems that are presently being commissioned at Los Alamos National Laboratory for very precise measurements of (n,{gamma}) and (n,f) cross-sections using small quantities of radioactive samples. DANCE (Detector for Advanced Neutron-Capture Experiments), a 4 {pi} gamma array made up of 160 BaF{sub 2} detectors, is designed to measure neutron capture cross-sections of unstable nuclei in the low-energy range (thermal to {approx}500 keV). The high granularity and high detection efficiency of DANCE, combined with the high TOF-neutron flux available at the Lujan Center provides a versatile tool for measuring many important cross section data using radioactive and isotopically enriched targets of about 1 milligram. Another powerful instrument is the Lead-slowing down spectrometer (LSDS), which will enable the measurement of neutron-induced fission cross-section of U-235m and other short-lived actinides in a energy range from 1-200 keV with sample sizes down to 10 nanograms. Due to the short half-life of the U-235m isomer (T{sub 1/2} = 26 minutes), the samples must be rapidly and repeatedly extracted from its {sup 239}Pu parent. Since {sup 239}Pu is itself highly fissile, the separation must not only be rapid, but must also be of very high purity (the Pu must be removed from the U with a decontamination factor >10{sup 12}). Once extracted and purified, the {sup 235m}U isomer would be electrodeposited on solar cells as a fission detector and placed within the LSDS for direct (n,f) cross section measurements. The

  17. Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers.

    PubMed

    Thomas, Rusha; Kim, Myoung H

    2007-02-01

    Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors. PMID:16924414

  18. Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans.

    PubMed

    Watson, Emma; Olin-Sandoval, Viridiana; Hoy, Michael J; Li, Chi-Hua; Louisse, Timo; Yao, Victoria; Mori, Akihiro; Holdorf, Amy D; Troyanskaya, Olga G; Ralser, Markus; Walhout, Albertha Jm

    2016-01-01

    Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild. PMID:27383050

  19. Infrared laser induced plasma diagnostics of silver target

    SciTech Connect

    Ahmat, L. Nadeem, Ali; Ahmed, I.

    2014-09-15

    In the present work, the optical emission spectra of silver (Ag) plasma have been recorded and analyzed using the laser induced breakdown spectroscopy technique. The emission line intensities and plasma parameters were investigated as a function of lens to sample distance, laser irradiance, and distance from the target surface. The electron number density (n{sub e}) and electron temperature (T{sub e}) were determined using the Stark broadened line profile and Boltzmann plot method, respectively. A gradual increase in the spectral line intensities and the plasma parameters, n{sub e} from 2.89 × 10{sup 17} to 3.92 × 10{sup 17 }cm{sup −3} and T{sub e} from 4662 to 8967 K, was observed as the laser irradiance was increased 2.29 × 10{sup 10}–1.06 × 10{sup 11} W cm{sup −2}. The spatial variations in n{sub e} and T{sub e} were investigated from 0 to 5.25 mm from the target surface, yielding the electron number density from 4.78 × 10{sup 17} to 1.72 × 10{sup 17 }cm{sup −3} and electron temperature as 9869–3789 K. In addition, the emission intensities and the plasma parameters of silver were investigated by varying the ambient pressure from 0.36 to 1000 mbars.

  20. Hypoxia inducible factor in hepatocellular carcinoma: A therapeutic target

    PubMed Central

    Lin, Daniel; Wu, Jennifer

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed and deadly cancers worldwide; its incidence has been rising in the United States due to the increase in hepatitis C associated cirrhosis and the growing epidemic of obesity. There have been no effective therapeutic options in the advanced disease setting beyond sorafenib, a multi-targeted tyrosine kinase inhibitor that showed significant survival benefit. Because of this, there is an urgent need to search for novel pathways in sorafenib experienced patients. This review will focus on the role of hypoxia and hypoxia-inducible factor alpha (HIF-1α) in cancer development, specifically in HCC. We will discuss the biology of HIF-1α, the pathways with which it interacts, and the function of HIF-1α in HCC. Furthermore, we will review studies highlighting the relevance of HIF-1α in the clinical setting, as well as the pre-clinical data supporting its further investigation. Finally, we will conclude with a discussion of the potential role of a HIF-1α mRNA antagonist for the treatment of HCC, and hypothesize the ways in which such an inhibitor may be best utilized in the management of advanced HCC. Hypoxia plays a significant role in the development of HCC. HIF-1α is a key transcription factor involved in the hypoxic response of cancer cells. It activates transcription of genes responsible for angiogenesis, glucose metabolism, proliferation, invasion and metastasis in HCC. Its involvement in multiple, essential tumor pathways makes it an attractive potential therapeutic target in HCC. PMID:26576101

  1. Hairpin DNA probes based on target-induced in situ generation of luminescent silver nanoclusters.

    PubMed

    Xiao, Yan; Wu, Zhengjun; Wong, Kwok-Yin; Liu, Zhihong

    2014-05-14

    Novel hairpin DNA probes are designed and constructed based on target-induced in situ generation of luminescent silver nanoclusters. This design allows specific and versatile detection of diverse targets with easy operation and low cost. PMID:24686790

  2. Multiple Targets for Drug-Induced Mitochondrial Toxicity.

    PubMed

    Wallace, Kendall B

    2015-01-01

    Mitochondrial toxicity is rapidly gaining the interest of researchers and practitioners as a prominent liability in drug discovery and development, accounting for a growing proportion of preclinical drug attrition and post-market withdrawals or black box warnings by the U.S. FDA. To date, the focus of registries of drugs that elicit mitochondrial toxicity has been largely restricted to those that either inhibit the mitochondrial electron transport chain (ETC) or uncouple mitochondrial oxidative phosphorylation. Less appreciated are the toxicities that are secondary to the drug affecting either the molecular regulation, assembly or incorporation of the ETC into the inner mitochondrial membrane or those that limit substrate availability. The current article describes the complexities of molecular events and biochemical pathways required to sustain mitochondrial fidelity and substrate homeostasis with examples of drugs that interfere which the various pathways. The principal objective of this review is to shed light on the broader scope of drug-induced mitochondrial toxicities and how these secondary targets may account for a large portion of drug failures. PMID:25973981

  3. Targeted approaches to induce immune tolerance for Pompe disease therapy.

    PubMed

    Doerfler, Phillip A; Nayak, Sushrusha; Corti, Manuela; Morel, Laurence; Herzog, Roland W; Byrne, Barry J

    2016-01-01

    Enzyme and gene replacement strategies have developed into viable therapeutic approaches for the treatment of Pompe disease (acid α-glucosidase (GAA) deficiency). Unfortunately, the introduction of GAA and viral vectors encoding the enzyme can lead to detrimental immune responses that attenuate treatment benefits and can impact patient safety. Preclinical and clinical experience in addressing humoral responses toward enzyme and gene therapy for Pompe disease have provided greater understanding of the immunological consequences of the provided therapy. B- and T-cell modulation has been shown to be effective in preventing infusion-associated reactions during enzyme replacement therapy in patients and has shown similar success in the context of gene therapy. Additional techniques to induce humoral tolerance for Pompe disease have been the targeted expression or delivery of GAA to discrete cell types or tissues such as the gut-associated lymphoid tissues, red blood cells, hematopoietic stem cells, and the liver. Research into overcoming preexisting immunity through immunomodulation and gene transfer are becoming increasingly important to achieve long-term efficacy. This review highlights the advances in therapies as well as the improved understanding of the molecular mechanisms involved in the humoral immune response with emphasis on methods employed to overcome responses associated with enzyme and gene therapies for Pompe disease. PMID:26858964

  4. Targeted approaches to induce immune tolerance for Pompe disease therapy

    PubMed Central

    Doerfler, Phillip A; Nayak, Sushrusha; Corti, Manuela; Morel, Laurence; Herzog, Roland W; Byrne, Barry J

    2016-01-01

    Enzyme and gene replacement strategies have developed into viable therapeutic approaches for the treatment of Pompe disease (acid α-glucosidase (GAA) deficiency). Unfortunately, the introduction of GAA and viral vectors encoding the enzyme can lead to detrimental immune responses that attenuate treatment benefits and can impact patient safety. Preclinical and clinical experience in addressing humoral responses toward enzyme and gene therapy for Pompe disease have provided greater understanding of the immunological consequences of the provided therapy. B- and T-cell modulation has been shown to be effective in preventing infusion-associated reactions during enzyme replacement therapy in patients and has shown similar success in the context of gene therapy. Additional techniques to induce humoral tolerance for Pompe disease have been the targeted expression or delivery of GAA to discrete cell types or tissues such as the gut-associated lymphoid tissues, red blood cells, hematopoietic stem cells, and the liver. Research into overcoming preexisting immunity through immunomodulation and gene transfer are becoming increasingly important to achieve long-term efficacy. This review highlights the advances in therapies as well as the improved understanding of the molecular mechanisms involved in the humoral immune response with emphasis on methods employed to overcome responses associated with enzyme and gene therapies for Pompe disease. PMID:26858964

  5. Rewiring Riboswitches to Create New Genetic Circuits in Bacteria.

    PubMed

    Robinson, C J; Medina-Stacey, D; Wu, M-C; Vincent, H A; Micklefield, J

    2016-01-01

    Riboswitches are RNA elements that control the expression of genes through a variety of mechanisms in response to the specific binding of small-molecule ligands. Since their discovery, riboswitches have shown promise for the artificial control of transcription or translation of target genes, be it for industrial biotechnology, protein expression, metabolic engineering, antimicrobial target validation, or gene function discovery. However, natural riboswitches are often unsuitable for these purposes due to their regulation by small molecules which are already present within the cell. For this reason, research has focused on creating riboswitches that respond to alternative biologically inert ligands or to molecules which are of interest for biosensing. Here we present methods for the development of artificial riboswitches in Gram-negative and Gram-positive bacteria. These methods are based on reengineering natural aptamers to change their ligand specificity toward molecules which do not bind the original aptamer (ie, that are orthogonal to the original). The first approach involves targeted mutagenesis of native riboswitches to change their specificity toward rationally designed synthetic ligand analogs. The second approach involves the fusion of previously validated orthogonal aptamers with native expression platforms to create novel chimeric riboswitches for the microbial target. We establish the applicability of these methods both for the control of exogenous genes as well as for the control of native genes. PMID:27417935

  6. Cullin E3 Ligases and Their Rewiring by Viral Factors

    PubMed Central

    Mahon, Cathal; Krogan, Nevan J.; Craik, Charles S.; Pick, Elah

    2014-01-01

    The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity. PMID:25314029

  7. Alcohol-induced ciliary dysfunction targets the outer dynein arm

    PubMed Central

    Yang, Fan; Pavlik, Jacqueline; Fox, Laura; Scarbrough, Chasity; Sale, Winfield S.; Sisson, Joseph H.

    2015-01-01

    Alcohol abuse results in an increased incidence of pulmonary infection, in part attributable to impaired mucociliary clearance. Analysis of motility in mammalian airway cilia has revealed that alcohol impacts the ciliary dynein motors by a mechanism involving altered axonemal protein phosphorylation. Given the highly conserved nature of cilia, it is likely that the mechanisms for alcohol-induced ciliary dysfunction (AICD) are conserved. Thus we utilized the experimental advantages offered by the model organism, Chlamydomonas, to determine the precise effects of alcohol on ciliary dynein activity and identify axonemal phosphoproteins that are altered by alcohol exposure. Analysis of live cells or reactivated cell models showed that alcohol significantly inhibits ciliary motility in Chlamydomonas via a mechanism that is part of the axonemal structure. Taking advantage of informative mutant cells, we found that alcohol impacts the activity of the outer dynein arm. Consistent with this finding, alcohol exposure results in a significant reduction in ciliary beat frequency, a parameter of ciliary movement that requires normal outer dynein arm function. Using mutants that lack specific heavy-chain motor domains, we have determined that alcohol impacts the β- and γ-heavy chains of the outer dynein arm. Furthermore, using a phospho-threonine-specific antibody, we determined that the phosphorylation state of DCC1 of the outer dynein arm-docking complex is altered in the presence of alcohol, and its phosphorylation correlates with AICD. These results demonstrate that alcohol targets specific outer dynein arm components and suggest that DCC1 is part of an alcohol-sensitive mechanism that controls outer dynein arm activity. PMID:25595647

  8. Studies of wave phenomena using HF-induced scatter target

    NASA Astrophysics Data System (ADS)

    Blagoveshchenskaya, N.; Borisova, T.; Kornienko, V.; Rietveld, M.; Frolov, V.; Uryadov, V.; Kagan, L.; Yampolski, Y.; Vertogradov, G.; Kelley, M.

    Experimental results from Tromso and Sura heating experiments at high and mid-latitudes are examined It was shown that the combination of HF-induced target and bi-static HF Doppler radio scatter observations is a profitable method for the identification and studies of wave phenomena of different origin We analysed the ULF activity in the Pc 3-4 range and the medium-scale traveling ionospheric disturbances TIDs at high and mid-latitudes Bi-static HF Doppler radio scatter observations were carried out on the London-Tromso-St Petersburg path in the course of Tromso heating experiments During Sura heating experiments multi-position bi-static HF Doppler radio scatter observations were simultaneously performed at three reception points including St Petersburg Kharkov and Rostov-on-Don Ray tracing and Doppler shift simulations were made for all experiments Parameters of ULF waves were found The interesting feature detected from Sura heating experiment was the dependence of the ULF wave parameters from the effective radiated power of the heating facility Medium-scale TIDs were observed in the evening and pre-midnight hours TIDs in the auroral E region with periods of 20-25 min were traveling southward at speeds from 190-250 m s TIDs in the mid-latitudinal F region with periods from 15 to 45 min were at speeds between 40 and 120 m s During quiet magnetic conditions the waves were traveling in the north-east direction In disturbed conditions the waves were moving in the south-west direction with higher speeds as compared with quiet conditions Possible mechanisms

  9. Epidermal Growth Factor Receptor-Targeted Photosensitizer Selectively Inhibits EGFR Signaling and Induces Targeted Phototoxicity In Ovarian Cancer Cells

    PubMed Central

    Abu-Yousif, Adnan O.; Moor, Anne C. E.; Zheng, Xiang; Savellano, Mark D.; Yu, Weiping; Selbo, Pål K.; Hasan, Tayyaba

    2012-01-01

    Targeted photosensitizer delivery to EGFR expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyin monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody. PMID:22266098

  10. Structural flexibility of intrinsically disordered proteins induces stepwise target recognition

    NASA Astrophysics Data System (ADS)

    Shirai, Nobu C.; Kikuchi, Macoto

    2013-12-01

    An intrinsically disordered protein (IDP) lacks a stable three-dimensional structure, while it folds into a specific structure when it binds to a target molecule. In some IDP-target complexes, not all target binding surfaces are exposed on the outside, and intermediate states are observed in their binding processes. We consider that stepwise target recognition via intermediate states is a characteristic of IDP binding to targets with "hidden" binding sites. To investigate IDP binding to hidden target binding sites, we constructed an IDP lattice model based on the HP model. In our model, the IDP is modeled as a chain and the target is modeled as a highly coarse-grained object. We introduced motion and internal interactions to the target to hide its binding sites. In the case of unhidden binding sites, a two-state transition between the free states and a bound state is observed, and we consider that this represents coupled folding and binding. Introducing hidden binding sites, we found an intermediate bound state in which the IDP forms various structures to temporarily stabilize the complex. The intermediate state provides a scaffold for the IDP to access the hidden binding site. We call this process multiform binding. We conclude that structural flexibility of IDPs enables them to access hidden binding sites and this is a functional advantage of IDPs.

  11. Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos

    PubMed Central

    Kong, E.Y.; Yeung, W.K.; Chan, T.K.Y.; Cheng, S.H.; Yu, K.N.

    2016-01-01

    The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies. PMID:27529238

  12. Nonlinear preferential rewiring in fixed-size networks as a diffusion process.

    PubMed

    Johnson, Samuel; Torres, Joaquín J; Marro, Joaquín

    2009-05-01

    We present an evolving network model in which the total numbers of nodes and edges are conserved, but in which edges are continuously rewired according to nonlinear preferential detachment and reattachment. Assuming power-law kernels with exponents alpha and beta , the stationary states which the degree distributions evolve toward exhibit a second-order phase transition-from relatively homogeneous to highly heterogeneous (with the emergence of starlike structures) at alpha=beta . Temporal evolution of the distribution in this critical regime is shown to follow a nonlinear diffusion equation, arriving at either pure or mixed power laws of exponents -alpha and 1-alpha . PMID:19518399

  13. Inducing Oncoprotein Degradation to Improve Targeted Cancer Therapy1

    PubMed Central

    Ray, Dipankar; Cuneo, Kyle C.; Rehemtulla, Alnawaz; Lawrence, Theodore S.; Nyati, Mukesh K.

    2015-01-01

    Over the past decade, inhibition of the kinase activities of oncogenic proteins using small molecules and antibodies has been a mainstay of our anticancer drug development effort, resulting in several Food and Drug Administration–approved cancer therapies. The clinical effectiveness of kinase-targeted agents has been inconsistent, mostly because of the development of resistance. The expression and function of oncoproteins and tumor suppressors are regulated by numerous posttranslational protein modifications including phosphorylation, ubiquitination, and acetylation; hence, targeting specific posttranslational protein modifications provides for an attractive strategy for anticancer drug development. The present review discusses the hypothesis that targeted degradation of an oncoprotein may overcome many of the shortcomings seen with kinase inhibitors and that the approach would enable targeted inhibition of oncogenic proteins previously thought to be undruggable. PMID:26476077

  14. Shock-induced perturbation evolution in planar laser targets

    NASA Astrophysics Data System (ADS)

    Aglitskiy, Y.; Karasik, M.; Velikovich, A. L.; Serlin, V.; Weaver, J. L.; Kessler, T. J.; Schmitt, A. J.; Obenschain, S. P.; Metzler, N.; Oh, J.

    2013-10-01

    Experimental studies of hydrodynamic perturbation evolution triggered by a laser-driven shock wave in a planar target done on the KrF Nike laser facility are reported. The targets were made of solid plastic and/or plastic foam with single mode sinusoidal perturbation on the front or back surface or plastic/foam interface. Two specific cases are discussed. When a planar solid plastic target rippled at the front side is irradiated with a 350 ps long laser pulse, ablative Richtmyer-Meshkov (RM) oscillation of its areal mass modulation amplitude is detected while the laser is on, followed by observed strong oscillations of the areal mass in the unsupported shock flow after the laser pulse ends. When the target is rippled at the rear side, the nature of the perturbation evolution after the shock breakout is determined by the strength of the laser-driven shock wave. At pressure below 1 Mbar shock interaction with rear-surface ripples produces planar collimated jets manifesting the development of a classical RM instability in a weakly compressible shocked fluid. At shock pressure ~ 8 Mbar sufficient for vaporizing the shocked target material we observed instead the strong areal mass oscillations characteristic of a rippled centered rarefaction wave. Work supported by US DOE, Defense Programs.

  15. Rewiring translation - Genetic code expansion and its applications.

    PubMed

    Neumann, Heinz

    2012-07-16

    With few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA(CUA) pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies. PMID:22710184

  16. Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

    PubMed Central

    Watson, Emma; Olin-Sandoval, Viridiana; Hoy, Michael J; Li, Chi-Hua; Louisse, Timo; Yao, Victoria; Mori, Akihiro; Holdorf, Amy D; Troyanskaya, Olga G; Ralser, Markus; Walhout, Albertha JM

    2016-01-01

    Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild. DOI: http://dx.doi.org/10.7554/eLife.17670.001 PMID:27383050

  17. Rewiring yeast sugar transporter preference through modifying a conserved protein motif

    PubMed Central

    Young, Eric M.; Tong, Alice; Bui, Hang; Spofford, Caitlin; Alper, Hal S.

    2014-01-01

    Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe38Ile39Met40, Scheffersomyces stipitis rgt2 Phe38 and Met40, and Saccharomyces cerevisiae hxt7 Ile39Met40Met340 all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties. PMID:24344268

  18. Rewiring yeast sugar transporter preference through modifying a conserved protein motif.

    PubMed

    Young, Eric M; Tong, Alice; Bui, Hang; Spofford, Caitlin; Alper, Hal S

    2014-01-01

    Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe(38)Ile(39)Met(40), Scheffersomyces stipitis rgt2 Phe(38) and Met(40), and Saccharomyces cerevisiae hxt7 Ile(39)Met(40)Met(340) all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties. PMID:24344268

  19. Radiation induced cavitation: A possible phenomenon in liquid targets?

    SciTech Connect

    West, C.D.

    1998-07-01

    The proposed design of a new, short-pulse spallation neutron source includes a liquid mercury target irradiated with a 1 GeV proton beam. This paper explores the possibility that cavitation bubbles may be formed in the mercury and briefly discusses some design features that could avoid harmful effects should cavitation take place.

  20. Flickering task–irrelevant distractors induce dilation of target duration depending upon cortical distance

    PubMed Central

    Okajima, Miku; Yotsumoto, Yuko

    2016-01-01

    Flickering stimuli are perceived to be longer than stable stimuli. This so-called “flicker-induced time dilation” has been investigated in a number of studies, but the factors critical for this effect remain unclear. We explored the spatial distribution of the flicker effect and examined how the flickering task-irrelevant distractors spatially distant from the target induce time dilation. In two experiments, we demonstrated that flickering distractors dilated the perceived duration of the target stimulus even though the target stimulus itself was stable. In addition, when the distractor duration was much longer than the target duration, a flickering distractor located ipsilateral to the target caused greater time dilation than did a contralateral distractor. Thus the amount of dilation depended on the distance between the cortical areas responsible for the stimulus locations. These findings are consistent with the recent study reporting that modulation of neural oscillators encoding the interval duration could explain flicker-induced time dilation. PMID:27577614

  1. Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes*

    PubMed Central

    Chen, Xi; Khambu, Bilon; Zhang, Hao; Gao, Wentao; Li, Min; Chen, Xiaoyun; Yoshimori, Tamotsu; Yin, Xiao-Ming

    2014-01-01

    Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62. PMID:24619419

  2. Hypoxia-inducible factors as molecular targets for liver diseases.

    PubMed

    Ju, Cynthia; Colgan, Sean P; Eltzschig, Holger K

    2016-06-01

    Liver disease is a growing global health problem, as deaths from end-stage liver cirrhosis and cancer are rising across the world. At present, pharmacologic approaches to effectively treat or prevent liver disease are extremely limited. Hypoxia-inducible factor (HIF) is a transcription factor that regulates diverse signaling pathways enabling adaptive cellular responses to perturbations of the tissue microenvironment. HIF activation through hypoxia-dependent and hypoxia-independent signals have been reported in liver disease of diverse etiologies, from ischemia-reperfusion-induced acute liver injury to chronic liver diseases caused by viral infection, excessive alcohol consumption, or metabolic disorders. This review summarizes the evidence for HIF stabilization in liver disease, discusses the mechanistic involvement of HIFs in disease development, and explores the potential of pharmacological HIF modifiers in the treatment of liver disease. PMID:27094811

  3. Holmium target fragmentation induced by intermediate energy /sup 12/C and /sup 16/O ions

    SciTech Connect

    Kraus, R.H. Jr.

    1986-01-01

    Target fragment (40 < A < 180) production cross sections were measured using off-line gamma-ray spectroscopy for the interaction of 208 MeV /sup 12/C, 272 MeV /sup 16/O, 442 MeV/sup 12/C, 1020 MeV /sup 12/C, and 1635 MeV /sup 16/O with /sup 165/Ho. Target fragment isobaric yields were deduced from these measurements. Trans-target nuclides were identified for all reaction systems. Nuclides up to 4 Z-units above the target were identified for 208 MeV /sup 12/C and 272 MeV /sup 16/O induced reactions, to 3 Z-units above the target for 442 MeV /sup 12/C and 1020 MeC /sup 12/C induced reactions, and to 2 Z-units above the target for 1635 MeV /sup 16/O induced reactions. Fission was observed to decrease between 17 MeV/A and 37 MeV/A from 13% of the reaction cross section to 4% for /sup 12/C induced reactions. No fission contribution was observed for 1020 MeV /sup 12/C and 1635 MeV /sup 16/O induced interactions.

  4. Regulatory circuit rewiring and functional divergence of the duplicate admp genes in dorsoventral axial patterning.

    PubMed

    Chang, Yi-Cheng; Pai, Chih-Yu; Chen, Yi-Chih; Ting, Hsiu-Chi; Martinez, Pedro; Telford, Maximilian J; Yu, Jr-Kai; Su, Yi-Hsien

    2016-02-01

    The spatially opposed expression of Antidorsalizing morphogenetic protein (Admp) and BMP signals controls dorsoventral (DV) polarity across Bilateria and hence represents an ancient regulatory circuit. Here, we show that in addition to the conserved admp1 that constitutes the ancient circuit, a second admp gene (admp2) is present in Ambulacraria (Echinodermata+Hemichordata) and two marine worms belonging to Xenoturbellida and Acoelomorpha. The phylogenetic distribution implies that the two admp genes were duplicated in the Bilaterian common ancestor and admp2 was subsequently lost in chordates and protostomes. We show that the ambulacrarian admp1 and admp2 are under opposite transcriptional control by BMP signals and knockdown of Admps in sea urchins impaired their DV polarity. Over-expression of either Admps reinforced BMP signaling but resulted in different phenotypes in the sea urchin embryo. Our study provides an excellent example of signaling circuit rewiring and protein functional changes after gene duplications. PMID:26719126

  5. Evolution. Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system.

    PubMed

    Taylor, Tiffany B; Mulley, Geraldine; Dills, Alexander H; Alsohim, Abdullah S; McGuffin, Liam J; Studholme, David J; Silby, Mark W; Brockhurst, Michael A; Johnson, Louise J; Jackson, Robert W

    2015-02-27

    A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homolog of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and toward its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps. PMID:25722415

  6. Molecular Principles of Gene Fusion Mediated Rewiring of Protein Interaction Networks in Cancer.

    PubMed

    Latysheva, Natasha S; Oates, Matt E; Maddox, Louis; Flock, Tilman; Gough, Julian; Buljan, Marija; Weatheritt, Robert J; Babu, M Madan

    2016-08-18

    Gene fusions are common cancer-causing mutations, but the molecular principles by which fusion protein products affect interaction networks and cause disease are not well understood. Here, we perform an integrative analysis of the structural, interactomic, and regulatory properties of thousands of putative fusion proteins. We demonstrate that genes that form fusions (i.e., parent genes) tend to be highly connected hub genes, whose protein products are enriched in structured and disordered interaction-mediating features. Fusion often results in the loss of these parental features and the depletion of regulatory sites such as post-translational modifications. Fusion products disproportionately connect proteins that did not previously interact in the protein interaction network. In this manner, fusion products can escape cellular regulation and constitutively rewire protein interaction networks. We suggest that the deregulation of central, interaction-prone proteins may represent a widespread mechanism by which fusion proteins alter the topology of cellular signaling pathways and promote cancer. PMID:27540857

  7. Rewiring neural circuits by the insertion of ectopic electrical synapses in transgenic C. elegans

    PubMed Central

    Rabinowitch, Ithai; Chatzigeorgiou, Marios; Zhao, Buyun; Treinin, Millet; Schafer, William R.

    2014-01-01

    Neural circuits are functional ensembles of neurons that are selectively interconnected by chemical or electrical synapses. Here we describe a synthetic biology approach to the study of neural circuits, whereby new electrical synapses can be introduced in novel sites in the neuronal circuitry to reprogram behaviour. We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt. Connecting these neurons by an ectopic electrical synapse led to a loss of lateral asymmetry and altered chemotaxis behaviour. In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal’s behavioural response to benzaldehyde. These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans. PMID:25026983

  8. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine.

    PubMed

    Qin, Jiufu; Zhou, Yongjin J; Krivoruchko, Anastasia; Huang, Mingtao; Liu, Lifang; Khoomrung, Sakda; Siewers, Verena; Jiang, Bo; Nielsen, Jens

    2015-01-01

    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l(-1) with a yield of 67 mg (g glucose)(-1) in shake-flask cultures and a titre of 5.1 g l(-1) in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals. PMID:26345617

  9. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

    PubMed Central

    Qin, Jiufu; Zhou, Yongjin J.; Krivoruchko, Anastasia; Huang, Mingtao; Liu, Lifang; Khoomrung, Sakda; Siewers, Verena; Jiang, Bo; Nielsen, Jens

    2015-01-01

    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l−1 with a yield of 67 mg (g glucose)−1 in shake-flask cultures and a titre of 5.1 g l−1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals. PMID:26345617

  10. Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

    PubMed

    Lee, Eric A; Angka, Leonard; Rota, Sarah-Grace; Hanlon, Thomas; Mitchell, Andrew; Hurren, Rose; Wang, Xiao Ming; Gronda, Marcela; Boyaci, Ezel; Bojko, Barbara; Minden, Mark; Sriskanthadevan, Shrivani; Datti, Alessandro; Wrana, Jeffery L; Edginton, Andrea; Pawliszyn, Janusz; Joseph, Jamie W; Quadrilatero, Joe; Schimmer, Aaron D; Spagnuolo, Paul A

    2015-06-15

    Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. PMID:26077472

  11. Targeted induction of endoplasmic reticulum stress induces cartilage pathology.

    PubMed

    Rajpar, M Helen; McDermott, Ben; Kung, Louise; Eardley, Rachel; Knowles, Lynette; Heeran, Mel; Thornton, David J; Wilson, Richard; Bateman, John F; Poulsom, Richard; Arvan, Peter; Kadler, Karl E; Briggs, Michael D; Boot-Handford, Raymond P

    2009-10-01

    Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS-causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF-mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress-inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix

  12. Alcohol-induced hypertension: an important healthcare target in Belgium.

    PubMed

    Collart, F; de Timary, P; Dom, G; Dor, B D; Duprez, D; Lengelé, J-P; Matthys, F; Peuskens, H; Rehm, J; Stärkel, P

    2015-12-01

    Excessive alcohol intake is one of the leading causes of premature death in Europe and particularly in Belgium. Belgian people are consuming more alcohol per year than the European average. It is well established that excessive alcohol consumption is a significant predictor of the development of hypertension (HTN). Two million adults in Belgium suffer from HTN and this number will increase to three million by 2025. Less than 50% of Belgian people treated for HTN are well-controlled. Alcohol reduction in patients with HTN can significantly lower systolic and diastolic blood pressure. After reviewing the epidemiology of HTN and alcohol disorders in Belgium, this paper will focus on the rationale for alcohol screening and brief intervention in primary care. It will also describe the barriers to alcohol screening, and what could be the benefits of alcohol screening for our healthcare system. The authors believe that early identification through alcohol screening and brief intervention in general practice can help to improve the management of patients with HTN, to reach the targets of the WHO Global Action Plan, i.e., a 25% relative reduction in the risk of premature mortality from cardiovascular diseases, cancer, diabetes or chronic respiratory diseases. They are also convinced that this would allow achieving major healthcare savings. PMID:26135944

  13. Initial observations of cavitation-induced erosion of liquid metal spallation target vessels at the Spallation Neutron Source

    SciTech Connect

    McClintock, David A; Riemer, Bernie; Ferguson, Phillip D; Carroll, Adam J; Dayton, Michael J

    2012-01-01

    During operation of the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory the mechanical properties of the AISI 316L target module are altered by high-energy neutron and proton radiation. The interior surfaces of the target vessel are also damaged by cavitation-induced erosion, which results from repetitive rapid heating of the liquid mercury by high-energy proton beam pulses. Until recently no observations of cavitation-induced erosion were possible for conditions prototypical to the SNS. Post irradiation examination (PIE) of the first and second operational SNS targets was performed to gain insight into the radiation-induced changes in mechanical properties of the 316L target material and the extent of cavitation-induced erosion to the target vessel inner surfaces. Observations of cavitation-induced erosion of the first and second operational SNS target modules are presented here, including images of the target vessel interiors and specimens removed from the target beam-entrance regions.

  14. Hot fusion-evaporation cross sections of 44Ca-induced reactions with lanthanide targets

    NASA Astrophysics Data System (ADS)

    Werke, T. A.; Mayorov, D. A.; Alfonso, M. C.; Tereshatov, E. E.; Folden, C. M.

    2015-11-01

    Background: Previously reported cross sections of 45Sc-induced reactions with lanthanide targets are much smaller than 48Ca-induced reactions on the same targets. 44Ca is one proton removed from 45Sc and could be used to produce nuclei with a relative neutron content between those produced in the 45Sc- and 48Ca-induced reactions. Purpose: As part of a systematic investigation of fusion-evaporation reactions, cross sections of 44Ca-induced reactions on lanthanide targets were measured. These results are compared to available data for 48Ca- and 45Sc-induced fusion-evaporation cross sections on the same lanthanide targets. Collectively, these data provide insight into the importance of the survival against fission of excited compound nuclei produced near spherical shell closures. Methods: A beam of 6+Ca at an energy of ≈5 MeV /u was delivered by the K500 superconducting cyclotron at the Cyclotron Institute at Texas A&M University. The desired evaporation residues were selected by the Momentum Achromat Recoil Spectrometer and identified via their characteristic α -decay energies. Excitation functions for the 44Ca+158Gd ,159Tb, and 162Dy reactions were measured at five or more energies each. A theoretical model was employed to study the fusion-evaporation process. Results: The 44Ca-induced reactions have x n cross sections that are two orders of magnitude larger than 45Sc-induced reactions but two orders of magnitude smaller than 48Ca-induced reactions on the same targets. Proton emission competes effectively with neutron emission for the 44Ca+159Tb and 162Dy reactions. The maximum 4 n cross sections in the 44Ca+158Gd ,159Tb, and 162Dy reactions were 2100 ± 230 ,230 ± 20 , and 130 ±20 μ b , respectively. The 44Ca+158Gd and 159Tb cross sections are in good agreement with the respective cross bombardments of 48Ca+154Gd and 45Sc+158Gd once differences in capture cross sections and compound nucleus formation probabilities are corrected for. Conclusions: Excitation

  15. Oxidative DNA damage caused by inflammation may link to stress-induced non-targeted effects

    PubMed Central

    Sprung, Carl N.; Ivashkevich, Alesia; Forrester, Helen B.; Redon, Christophe E.; Georgakilas, Alexandros; Martin, Olga A.

    2013-01-01

    A spectrum of radiation-induced non-targeted effects has been reported during the last two decades since Nagasawa and Little first described a phenomenon in cultured cells that was later called the “bystander effect”. These non-targeted effects include radiotherapy-related abscopal effects, where changes in organs or tissues occur distant from the irradiated region. The spectrum of non-targeted effects continue to broaden over time and now embrace many types of exogenous and endogenous stressors that induce a systemic genotoxic response including a widely studied tumor microenvironment. Here we discuss processes and factors leading to DNA damage induction in non-targeted cells and tissues and highlight similarities in the regulation of systemic effects caused by different stressors. PMID:24041866

  16. Infrared small target detection via line-based reconstruction and entropy-induced suppression

    NASA Astrophysics Data System (ADS)

    Shang, Ke; Sun, Xiao; Tian, Jinwen; Li, Yansheng; Ma, Jiayi

    2016-05-01

    This paper proposes a novel infrared small target detection method which is composed of two stages. The first stage is implemented by line-based reconstruction for suppressing the background clutter, and the second stage is induced by information entropy for further standing out the targets. Compared with the state-of-the-art approaches, the proposed approach is able to achieve better performance in terms of efficiency and accuracy.

  17. Isospin effects on fragmentation in the asymmetric reactions induced by neutron-rich targets

    NASA Astrophysics Data System (ADS)

    Sharma, Arun

    2016-05-01

    To understand the isospin effects in terms of fragment's yield in the asymmetric reactions induced by neutron-rich targets, we perform a theoretical study using isospin-dependent quantum molecular dynamics (IQMD) model. Simulations are carried out for reactions of 16O+Br80,84,92 and 16O+Ag108,113,122. We envision that fragments's yield in the asymmetric collisions induced by neutron-rich targets is better candidate to study isospin effects via symmetry energy and nucleon-nucleon (nn) cross-sections. Also, pronounced effects of symmetry energy and cross-sections can be found at lower and higher beam energies, respectively.

  18. Infrared laser-induced gene expression in targeted single cells of Caenorhabditis elegans.

    PubMed

    Suzuki, Motoshi; Toyoda, Naoya; Shimojou, Masaki; Takagi, Shin

    2013-05-01

    Since the dawn of transgenic technology some 40 years ago, biologists have sought ways to manipulate, at their discretion, the expression of particular genes of interest in living organisms. The infrared laser-evoked gene operator (IR-LEGO) is a recently developed system for inducing gene expression in living organisms in a targeted fashion. It exploits the highly efficient capacity of an infrared laser for heating cells, to provide a high level of gene expression driven by heat-inducible promoters. By irradiating living specimens with a laser under a microscope, heat shock responses can be induced in individual cells, thereby inducing a particular gene, under the control of a heat shock promoter, in specifically targeted cells. In this review we first summarize previous attempts to drive transgene expression in organisms by using heat shock promoters, and then introduce the basic principle of the IR-LEGO system, and its applications. PMID:23614811

  19. Rewiring neuronal microcircuits of the brain via spine head protrusions--a role for synaptopodin and intracellular calcium stores.

    PubMed

    Verbich, David; Becker, Denise; Vlachos, Andreas; Mundel, Peter; Deller, Thomas; McKinney, R Anne

    2016-01-01

    Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient. PMID:27102112

  20. Validation of Laser-Induced Fluorescent Photogrammetric Targets on Membrane Structures

    NASA Technical Reports Server (NTRS)

    Jones, Thomas W.; Dorrington, Adrian A.; Shortis, Mark R.; Hendricks, Aron R.

    2004-01-01

    The need for static and dynamic characterization of a new generation of inflatable space structures requires the advancement of classical metrology techniques. A new photogrammetric-based method for non-contact ranging and surface profiling has been developed at NASA Langley Research Center (LaRC) to support modal analyses and structural validation of this class of space structures. This full field measurement method, known as Laser-Induced Fluorescence (LIF) photogrammetry, has previously yielded promising experimental results. However, data indicating the achievable measurement precision had not been published. This paper provides experimental results that indicate the LIF-photogrammetry measurement precision for three different target types used on a reflective membrane structure. The target types were: (1) non-contact targets generated using LIF, (2) surface attached retro-reflective targets, and (3) surface attached diffuse targets. Results from both static and dynamic investigations are included.

  1. Investigation of apoptotic events at molecular level induced by SERS guided targeted theranostic nanoprobe.

    PubMed

    Narayanan, Nisha; Nair, Lakshmi V; Karunakaran, Varsha; Joseph, Manu M; Nair, Jyothi B; N, Ramya A; Jayasree, Ramapurath S; Maiti, Kaustabh Kumar

    2016-06-01

    Herein, we have examined distinctive structural and functional variations of cellular components during apoptotic cell death induced by a targeted theranostic nanoprobe, MMP-SQ@GNR@LAH-DOX, which acted as a SERS "on/off" probe in the presence of a MMP protease and executed synergistic photothermal chemotherapy, as reflected by the SERS fingerprinting, corresponding to the phosphodiester backbone of DNA. PMID:27211810

  2. Autapse-induced target wave, spiral wave in regular network of neurons

    NASA Astrophysics Data System (ADS)

    Qin, HuiXin; Ma, Jun; Wang, ChunNi; Chu, RunTong

    2014-10-01

    Autapse is a type of synapse that connects axon and dendrites of the same neuron, and the effect is often detected by close-loop feedback in axonal action potentials to the owned dendritic tree. An artificial autapse was introduced into the Hindmarsh-Rose neuron model, and a regular network was designed to detect the regular pattern formation induced by autapse. It was found that target wave emerged in the network even when only a single autapse was considered. By increasing the (autapse density) number of neurons with autapse, for example, a regular area (2×2, 3×3, 4×4, 5×5 neurons) under autapse induced target wave by selecting the feedback gain and time-delay in autapse. Spiral waves were also observed under optimized feedback gain and time delay in autapses because of coherence-like resonance in the network induced by some electric autapses connected to some neurons. This confirmed that the electric autapse has a critical role in exciting and regulating the collective behaviors of neurons by generating stable regular waves (target waves, spiral waves) in the network. The wave length of the induced travelling wave (target wave, spiral wave), because of local effect of autapse, was also calculated to understand the waveprofile in the network of neurons.

  3. Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway

    PubMed Central

    Wan, Gang; Xie, Weidong; Liu, Zhenyan; Xu, Wei; Lao, Yuanzhi; Huang, Nunu; Cui, Kai; Liao, Meijian; He, Jie; Jiang, Yuyang; Yang, Burton B; Xu, Hongxi; Xu, Naihan; Zhang, Yaou

    2014-01-01

    Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3′ untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G1/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway. PMID:24262949

  4. Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase

    PubMed Central

    Richter, Erik; Ventz, Katharina; Harms, Manuela; Mostertz, Jörg; Hochgräfe, Falko

    2016-01-01

    Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease. PMID:27066479

  5. Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

    PubMed Central

    Faruqi, A F; Seidman, M M; Segal, D J; Carroll, D; Glazer, P M

    1996-01-01

    Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy. PMID:8943337

  6. Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach

    PubMed Central

    Cambronne, Xiaolu A.; Shen, Rongkun; Auer, Paul L.; Goodman, Richard H.

    2012-01-01

    Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA– RNA-induced silencing complex (RISC)–messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs. PMID:23184980

  7. Potent degradation of neuronal miRNAs induced by highly complementary targets

    PubMed Central

    de la Mata, Manuel; Gaidatzis, Dimos; Vitanescu, Mirela; Stadler, Michael B; Wentzel, Corinna; Scheiffele, Peter; Filipowicz, Witold; Großhans, Helge

    2015-01-01

    MicroRNAs (miRNAs) regulate target mRNAs by silencing them. Reciprocally, however, target mRNAs can also modulate miRNA stability. Here, we uncover a remarkable efficacy of target RNA-directed miRNA degradation (TDMD) in rodent primary neurons. Coincident with degradation, and while still bound to Argonaute, targeted miRNAs are 3′ terminally tailed and trimmed. Absolute quantification of both miRNAs and their decay-inducing targets suggests that neuronal TDMD is multiple turnover and does not involve co-degradation of the target but rather competes with miRNA-mediated decay of the target. Moreover, mRNA silencing, but not TDMD, relies on cooperativity among multiple target sites to reach high efficacy. This knowledge can be harnessed for effective depletion of abundant miRNAs. Our findings bring insight into a potent miRNA degradation pathway in primary neurons, whose TDMD activity greatly surpasses that of non-neuronal cells and established cell lines. Thus, TDMD may be particularly relevant for miRNA regulation in the nervous system. PMID:25724380

  8. A target-induced fluorescent nanoparticle for in situ monitoring of Zn(II).

    PubMed

    John, Carrie L; Huan, Yanfu; Wu, Xu; Jin, Yuhui; Pierce, David T; Zhao, Julia Xiaojun

    2013-09-01

    A target-induced fluorescent silica nanoparticle has been developed for the identification, enrichment and in situ determination of trace amounts of zinc(II). The nanoparticle combines the advantages of target-induced fluorescent compounds and the small size of the nanomaterial to produce a new, smarter nanosignaling material that is capable of selectively enriching a target and detecting a specific binding process in one step. As the target analyte, Zn(II), changes the fluorescence characteristics of the nanoparticle and effectively 'turns on' the fluorescence signal, no separation step is needed to confirm or quantify the binding process. The designed nanoparticle was characterized by several aspects prior to monitoring of Zn(II) in situ. The interferences from common metal ions were studied in detail. The photostability and reversibility of the sensing materials were investigated as well. The ability of this nanoparticle to detect the target Zn(II) provides a great advantage for in situ monitoring targets in biological samples under the fluorescence microscope. PMID:23799230

  9. Pictorial target control of schedule-induced attack in White Carneaux pigeons1

    PubMed Central

    Looney, Thomas A.; Cohen, Perrin S.

    1974-01-01

    Three pigeons with a history of attacking a mirror target, and two of six pigeons with no prior exposure to targets, attacked a colored photograph of a conspecific during exposure to intermittent schedules of reinforcement for key pecking. Rate of attack on the photograph decreased when the reinforcement schedule was removed. The topography, temporal pattern, and locus of attack on the picture were comparable to schedule-induced attack on live, stuffed, and mirror targets. When silhouette, outline, and plain paper targets were used, schedule-induced attack was more sensitive to a change in target characteristics with a concurrent target-preference procedure than with an analogous successive-testing procedure. The combined results of the two testing procedures indicated that an “upright” white-on-black silhouette of a pigeon with or without an eye was more effective in controlling attack than was a comparable “inverted” silhouette, an outline of a pigeon, or a piece of colored paper. PMID:16811765

  10. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2009-10-01

    Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells. PMID:19726585

  11. The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

    PubMed Central

    Shen, Hong Ming; Poirier, Michael G.; Allen, Michael J.; North, Justin; Lal, Ratnesh; Widom, Jonathan

    2009-01-01

    The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes. PMID:19380635

  12. Targeting ornithine decarboxylase in Myc-induced lymphomagenesis prevents tumor formation.

    PubMed

    Nilsson, Jonas A; Keller, Ulrich B; Baudino, Troy A; Yang, Chunying; Norton, Sara; Old, Jennifer A; Nilsson, Lisa M; Neale, Geoffrey; Kramer, Debora L; Porter, Carl W; Cleveland, John L

    2005-05-01

    Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention. PMID:15894264

  13. Tuning fresh: radiation through rewiring of central metabolism in streamlined bacteria.

    PubMed

    Eiler, Alexander; Mondav, Rhiannon; Sinclair, Lucas; Fernandez-Vidal, Leyden; Scofield, Douglas G; Schwientek, Patrick; Martinez-Garcia, Manuel; Torrents, David; McMahon, Katherine D; Andersson, Siv Ge; Stepanauskas, Ramunas; Woyke, Tanja; Bertilsson, Stefan

    2016-08-01

    Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner-Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype. PMID:26784354

  14. Search for the Heisenberg spin glass on rewired square lattices with antiferromagnetic interaction

    NASA Astrophysics Data System (ADS)

    Surungan, Tasrief; Bansawang B., J.; Tahir, Dahlang

    2016-03-01

    Spin glass (SG) is a typical magnetic system with frozen random spin orientation at low temperatures. The system exhibits rich physical properties, such as infinite number of ground states, memory effect, and aging phenomena. There are two main ingredients considered to be pivotal for the existence of SG behavior, namely, frustration and randomness. For the canonical SG system, frustration is led by the presence of competing interaction between ferromagnetic (FM) and antiferromagnetic (AF) couplings. Previously, Bartolozzi et al. [Phys. Rev. B73, 224419 (2006)], reported the SG properties of the AF Ising spins on scale free network (SFN). It is a new type of SG, different from the canonical one which requires the presence of both FM and AF couplings. In this new system, frustration is purely caused by the topological factor and its randomness is related to the irregular connectvity. Recently, Surungan et. al. [Journal of Physics: Conference Series, 640, 012001 (2015)] reported SG bahavior of AF Heisenberg model on SFN. We further investigate this type of system by studying an AF Heisenberg model on rewired square lattices. We used Replica Exchange algorithm of Monte Carlo Method and calculated the SG order parameter to search for the existence of SG phase.

  15. From Neurons to Neuron Neighborhoods: the Rewiring of the Cerebellar Cortex in Essential Tremor

    PubMed Central

    2014-01-01

    Remarkably little has been written on the biology of essential tremor (ET), despite its high prevalence. The olivary model, first proposed in the 1970s, is the traditional disease model for ET; however, the model is problematic for a number of reasons. Recently, intensive tissue-based studies have identified a series of structural changes in the brains of most ET cases, and nearly all of the observed changes are located in the cerebellar cortex. These studies suggest that Purkinje cells are central to the pathogenesis of ET and may thus provide a focus for the development of novel therapeutic strategies. Arising from these studies, a new model of ET proposes that the population of Purkinje cells represents the site of the initial molecular/cellular events leading to ET. Furthermore, a number of secondary changes/remodeling observed in the molecular and granular layers (i.e., in the Purkinje cell “neighborhood”) are likely to be of additional mechanistic importance. On a physiological level, the presence of remodeling indicates the likely formation of aberrant synapses and the creation of new/abnormal cortical circuits in ET. Specific efforts need to be devoted to understanding the cascade of biochemical and cellular events occurring in the Purkinje cell layer in ET and its neuron neighborhood, as well as the physiological effects of secondary remodeling/rewiring that are likely to be occurring in this brain region in ET. PMID:24435423

  16. Oxidative metabolism involved in non-targeted effects induced by proton radiation in intact Arabidopsis seeds.

    PubMed

    Mei, Tao; Yang, Gen; Quan, Yi; Wang, Weikang; Zhang, Weiming; Xue, Jianming; Wu, Lijun; Gu, Hongya; Schettino, Giuseppe; Wang, Yugang

    2011-01-01

    Non-targeted effects induced by ionizing radiation have been demonstrated both in vitro and in vivo. Previously, we have also demonstrated the existence of non-targeted effects in intact Arabidopsis seeds following low-energy heavy-ion radiation. In the present study, 6.5 MeV protons with 8 × 10(11) ions/cm(2) and 2 × 10(11) ions/cm(2) fluence respectively were used to irradiate non-shielded or partial-shielded Arabidopsis seeds to further explore the mechanisms which regulate in vivo non-targeted effects and to investigate the difference between damage caused by non-targeted effects and direct irradiation. Results showed that excess reactive oxygen species (ROS) are present in the non-irradiated part of the partially irradiated samples, indicating that in vivo non-targeted effects can promote the generation of excess metabolic ROS in the non-irradiated shoot apical meristem/root apical meristem cells. Furthermore, pretreatment with 0.5% ROS scavenger dimethyl sulfoxide (DMSO) or 0.02 mM reactive nitrogen species (RNS) scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) significantly suppresses the non-targeted effects in the partially irradiated samples, while in the whole-body irradiated samples, the cPTIO pretreatment has no effect. On the other hand using antioxidant enzyme assays, superoxide dismutase activity was found to increase for partial irradiated samples and decrease for the whole-body exposed seeds. Taken together, these results implicate that damage caused by non-targeted effects is different from that induced by direct irradiation in vivo. Metabolic products such as ROS and RNS are involved in the in vivo non-targeted effects. PMID:21343677

  17. The nucleus is the target for radiation-induced chromosomal instability

    NASA Technical Reports Server (NTRS)

    Kaplan, M. I.; Morgan, W. F.

    1998-01-01

    We have previously described chromosomal instability in cells of a human-hamster hybrid cell line after exposure to X rays. Chromosomal instability in these cells is characterized by the appearance of novel chromosomal rearrangements multiple generations after exposure to ionizing radiation. To identify the cellular target(s) for radiation-induced chromosomal instability, cells were treated with 125I-labeled compounds and frozen. Radioactive decays from 125I cause damage to the cell primarily at the site of their decay, and freezing the cells allows damage to accumulate in the absence of other cellular processes. We found that the decay of 125I-iododeoxyuridine, which is incorporated into the DNA, caused chromosomal instability. While cell killing and first-division chromosomal rearrangements increased with increasing numbers of 125I decays, the frequency of chromosomal instability was independent of dose. Chromosomal instability could also be induced from incorporation of 125I-iododeoxyuridine without freezing the cells for accumulation of decays. This indicates that DNA double-strand breaks in frozen cells resulting from 125I decays failed to lead to instability. Incorporation of an 125I-labeled protein (125I-succinyl-concanavalin A), which was internalized into the cell and/or bound to the plasma membrane, neither caused chromosomal instability nor potentiated chromosomal instability induced by 125I-iododeoxyuridine. These results show that the target for radiation-induced chromosomal instability in these cells is the nucleus.

  18. Radiation Induced Non-targeted Response: Mechanism and Potential Clinical Implications

    PubMed Central

    Hei, Tom K.; Zhou, Hongning; Chai, Yunfei; Ponnaiya, Brian; Ivanov, Vladimir N.

    2012-01-01

    Generations of students in radiation biology have been taught that heritable biological effects require direct damage to DNA. Radiation-induced non-targeted/bystander effects represent a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the biological consequences of exposure to low doses of radiation. Although radiation induced bystander effects have been well documented in a variety of biological systems, including 3D human tissue samples and whole organisms, the mechanism is not known. There is recent evidence that the NF-κB-dependent gene expression of interleukin 8, interleukin 6, cyclooxygenase-2, tumor necrosis factor and interleukin 33 in directly irradiated cells produced the cytokines and prostaglandin E2 with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. The observations that heritable DNA alterations can be propagated to cells many generations after radiation exposure and that bystander cells exhibit genomic instability in ways similar to directly hit cells indicate that the low dose radiation response is a complex interplay of various modulating factors. The potential implication of the non-targeted response in radiation induced secondary cancer is discussed. A better understanding of the mechanism of the non-targeted effects will be invaluable to assess its clinical relevance and ways in which the bystander phenomenon can be manipulated to increase therapeutic gain in radiotherapy. PMID:21143185

  19. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

    PubMed Central

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-01-01

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation. PMID:26389953

  20. Mitochondrial-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness

    PubMed Central

    Powers, Scott K.; Hudson, Matthew B.; Nelson, W. Bradley; Talbert, Erin E.; Min, Kisuk; Szeto, Hazel H.; Kavazis, Andreas N.; Smuder, Ashley J.

    2015-01-01

    BACKGROUND Mechanical ventilation (MV) is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged MV is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during MV, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of MV-induced reactive oxygen species (ROS) production in the diaphragm is important. OBJECTIVE These experiments tested the hypothesis that elevated mitochondrial ROS emission is required for MV-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm. DESIGN Cause and effect was determined by preventing MV-induced mitochondrial ROS emission in the diaphragm of rats using a novel mitochondrial-targeted antioxidant (SS-31). MEASUREMENTS AND MAIN RESULTS Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against MV-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against MV-induced myofiber atrophy and contractile dysfunction. CONCLUSIONS These results reveal that prevention of MV-induced increases in diaphragmatic mitochondrial ROS emission protects the diaphragm MV-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of ROS production in the diaphragm during prolonged MV. These results could lead to the development of a therapeutic intervention to impede MV-induced diaphragmatic weakness. PMID:21460706

  1. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel.

    PubMed

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-09-01

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, OPEN ACCESS Toxins 2015, 7 3672 BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation. PMID:26389953

  2. Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

    PubMed

    Bocharov, Alexander V; Wu, Tinghuai; Baranova, Irina N; Birukova, Anna A; Sviridov, Denis; Vishnyakova, Tatyana G; Remaley, Alan T; Eggerman, Thomas L; Patterson, Amy P; Birukov, Konstantin G

    2016-07-15

    Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema. PMID:27316682

  3. Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

    PubMed

    Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

    2016-02-01

    The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD

  4. Laser-induced synthesis and decay of Tritium under exposure of solid targets in heavy water

    NASA Astrophysics Data System (ADS)

    Barmina, E. V.; Timashev, S. F.; Shafeev, G. A.

    2016-03-01

    The processes of laser-assisted synthesis of Tritium nuclei and their laser-induced decay in cold plasma in the vicinity of solid targets (Au, Ti, Se, etc.) immersed into heavy water are experimentally realized at peak laser intensity of 1010-1013 W/cm2. Initial stages of Tritium synthesis and their laser-induced beta-decay are interpreted on the basis of non-elastic interaction of plasma electrons having kinetic energy of 5-10 eV with nuclei of Deuterium and Tritium, respectively.

  5. Flux and dose transmission through concrete of neutrons from proton induced reactions on various target elements

    NASA Astrophysics Data System (ADS)

    Maiti, Moumita; Nandy, Maitreyee; Roy, S. N.; Sarkar, P. K.

    2004-12-01

    Simple empirical expressions for transmission of flux and dose through concrete are presented for neutrons from proton induced reactions. For this purpose the neutron emission from different targets in proton induced reactions in the energy range 25-200 MeV have been considered. The calculated effective dose outside a concrete shield shows overall good agreement with the effective dose estimated from measured neutron flux in the framework of the Moyer model. The calculated effective attenuation length shows a rising trend with incident proton energy and shield thickness.

  6. Hispidulin induces mitochondrial apoptosis in acute myeloid leukemia cells by targeting extracellular matrix metalloproteinase inducer

    PubMed Central

    Gao, Hui; Liu, Yongji; Li, Kan; Wu, Tianhui; Peng, Jianjun; Jing, Fanbo

    2016-01-01

    Acute myeloid leukemia (AML) represents a heterogeneous group of hematological neoplasms with marked heterogeneity in response to both standard therapy and survival. Hispidulin, a flavonoid compound that is anactive ingredient in the traditional Chinese medicinal herb Salvia plebeia R. Br, has recently been reported to have anantitumor effect against solid tumors in vitro and in vivo. The aim of the present study was to investigate the effects of hispidulin on the human leukemia cell line in vitro and the underlying mechanisms of its actions on these cells. Our results showed that hispidulin inhibits AML cell proliferation in a dose- and time-dependent manner, and induces cell apoptosis throughan intrinsic mitochondrial pathway. Our results also revealed that hispidulin treatment significantly inhibits extracellular matrix metalloproteinase inducer (EMMPRIN) expression in both tested AML cell lines in a dose-dependent manner, and that the overexpression of EMMPRIN protein markedly attenuates hispidulin-induced cell apoptosis. Furthermore, our results strongly indicated that the modulating effect of hispidulin on EMMPRIN is correlated with its inhibitory effect on both the Akt and STAT3 signaling pathways. PMID:27158398

  7. Pivotal response treatment prompts a functional rewiring of the brain among individuals with autism spectrum disorder.

    PubMed

    Venkataraman, Archana; Yang, Daniel Y-J; Dvornek, Nicha; Staib, Lawrence H; Duncan, James S; Pelphrey, Kevin A; Ventola, Pamela

    2016-09-28

    Behavioral interventions for autism have gained prominence in recent years; however, the neural-systems-level targets of these interventions remain poorly understood. We use a novel Bayesian framework to extract network-based differences before and after a 16-week pivotal response treatment (PRT) regimen. Our results suggest that the functional changes induced by PRT localize to the posterior cingulate and are marked by a shift in connectivity from the orbitofrontal cortex to the occipital-temporal cortex. Our results illuminate a potential PRT-induced learning mechanism, whereby the neural circuits involved during social perception shift from sensory and attentional systems to higher-level object and face processing areas. PMID:27532879

  8. A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation.

    PubMed

    Reshat, Reshat; Priestley, Catherine C; Gooderham, Nigel J

    2012-11-01

    Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity. PMID:22914677

  9. Targeting the hallmarks of cancer with therapy-induced endoplasmic reticulum (ER) stress

    PubMed Central

    Garg, Abhishek D; Maes, Hannelore; van Vliet, Alexander R; Agostinis, Patrizia

    2015-01-01

    The endoplasmic reticulum (ER) is at the center of a number of vital cellular processes such as cell growth, death, and differentiation, crosstalk with immune or stromal cells, and maintenance of proteostasis or homeostasis, and ER functions have implications for various pathologies including cancer. Recently, a number of major hallmarks of cancer have been delineated that are expected to facilitate the development of anticancer therapies. However, therapeutic induction of ER stress as a strategy to broadly target multiple hallmarks of cancer has been seldom discussed despite the fact that several primary or secondary ER stress-inducing therapies have been found to exhibit positive clinical activity in cancer patients. In the present review we provide a brief historical overview of the major discoveries and milestones in the field of ER stress biology with important implications for anticancer therapy. Furthermore, we comprehensively discuss possible strategies enabling the targeting of multiple hallmarks of cancer with therapy-induced ER stress. PMID:27308392

  10. Investigation of apoptotic events at molecular level induced by SERS guided targeted theranostic nanoprobe

    NASA Astrophysics Data System (ADS)

    Narayanan, Nisha; Nair, Lakshmi V.; Karunakaran, Varsha; Joseph, Manu M.; Nair, Jyothi B.; N, Ramya A.; Jayasree, Ramapurath S.; Maiti, Kaustabh Kumar

    2016-06-01

    Herein, we have examined distinctive structural and functional variations of cellular components during apoptotic cell death induced by a targeted theranostic nanoprobe, MMP-SQ@GNR@LAH-DOX, which acted as a SERS ``on/off'' probe in the presence of a MMP protease and executed synergistic photothermal chemotherapy, as reflected by the SERS fingerprinting, corresponding to the phosphodiester backbone of DNA.Herein, we have examined distinctive structural and functional variations of cellular components during apoptotic cell death induced by a targeted theranostic nanoprobe, MMP-SQ@GNR@LAH-DOX, which acted as a SERS ``on/off'' probe in the presence of a MMP protease and executed synergistic photothermal chemotherapy, as reflected by the SERS fingerprinting, corresponding to the phosphodiester backbone of DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr03385g

  11. Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

    PubMed Central

    Crane, Ana M.; Kramer, Philipp; Bui, Jacquelin H.; Chung, Wook Joon; Li, Xuan Shirley; Gonzalez-Garay, Manuel L.; Hawkins, Finn; Liao, Wei; Mora, Daniela; Choi, Sangbum; Wang, Jianbin; Sun, Helena C.; Paschon, David E.; Guschin, Dmitry Y.; Gregory, Philip D.; Kotton, Darrell N.; Holmes, Michael C.; Sorscher, Eric J.; Davis, Brian R.

    2015-01-01

    Summary Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells. PMID:25772471

  12. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

    PubMed

    Crane, Ana M; Kramer, Philipp; Bui, Jacquelin H; Chung, Wook Joon; Li, Xuan Shirley; Gonzalez-Garay, Manuel L; Hawkins, Finn; Liao, Wei; Mora, Daniela; Choi, Sangbum; Wang, Jianbin; Sun, Helena C; Paschon, David E; Guschin, Dmitry Y; Gregory, Philip D; Kotton, Darrell N; Holmes, Michael C; Sorscher, Eric J; Davis, Brian R

    2015-04-14

    Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells. PMID:25772471

  13. Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway.

    PubMed

    Fu, Ying-Zi; Yan, Yuan-Yuan; He, Miao; Xiao, Qing-Huan; Yao, Wei-Fan; Zhao, Lin; Wu, Hui-Zhe; Yu, Zhao-Jin; Zhou, Ming-Yi; Lv, Mu-Tian; Zhang, Shan-Shan; Chen, Jian-Jun; Wei, Min-Jie

    2016-02-01

    Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs. PMID:26718029

  14. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    Lu, Wei; Han, Dai-Shu; Yuan, Ju; Andrieu, Jean-Marie

    1994-03-01

    Quantitative analysis of polymerase chain reaction (PCR) amplified HIV-1 DNA or cDNA fragments is attained using an automated system that combines capillary-gel electrophoresis (CGE) for high-efficiency separation and laser-induced fluorescence (LIF) for high-sensitivity detection. This system enables the detection of PCR-amplified multiple target DNA or cDNA in the same tube by a single injection with high precision.

  15. How to Target Activated Ras Proteins: Direct Inhibition vs. Induced Mislocalization.

    PubMed

    Brock, Ethan J; Ji, Kyungmin; Reiners, John J; Mattingly, Raymond R

    2016-01-01

    Oncogenic Ras proteins are a driving force in a significant set of human cancers and wildtype, unmutated Ras proteins likely contribute to the malignant phenotype of many more. The overall challenge of targeting activated Ras proteins has great promise to treat cancer, but this goal has yet to be achieved. Significant efforts and resources have been committed to inhibiting Ras, but these energies have so far made little impact in the clinic. Direct attempts to target activated Ras proteins have faced many obstacles, including the fundamental nature of the gain-of-function oncogenic activity being produced by a loss-of-function at the biochemical level. Nevertheless, there has been very promising recent pre-clinical progress. The major strategy that has so far reached the clinic aimed to inhibit activated Ras indirectly through blocking its post-translational modification and inducing its mislocalization. While these efforts to indirectly target Ras through inhibition of farnesyl transferase (FTase) were rationally designed, this strategy suffered from insufficient attention to the distinctions between the isoforms of Ras. This led to subsequent failures in large-scale clinical trials targeting K-Ras driven lung, colon, and pancreatic cancers. Despite these setbacks, efforts to indirectly target activated Ras through inducing its mislocalization have persisted. It is plausible that FTase inhibitors may still have some utility in the clinic, perhaps in combination with statins or other agents. Alternative approaches for inducing mislocalization of Ras through disruption of its palmitoylation cycle or interaction with chaperone proteins are in early stages of development. PMID:26423696

  16. Spectral selective radio frequency emissions from laser induced breakdown of target materials

    SciTech Connect

    Vinoth Kumar, L.; Manikanta, E.; Leela, Ch.; Prem Kiran, P.

    2014-08-11

    The radio frequency emissions scanned over broad spectral range (30 MHz–1 GHz) from single shot nanosecond (7 ns) and picosecond (30 ps) laser induced breakdown (LIB) of different target materials (atmospheric air, aluminum, and copper) are presented. The dominant emissions from ns-LIB, compared to those from the ps-LIB, indicate the presence and importance of atomic and molecular clusters in the plasma. The dynamics of laser pulse-matter interaction and the properties of the target materials were found to play an important role in determining the plasma parameters which subsequently determine the emissions. Thus, with a particular laser and target material, the emissions were observed to be spectral selective. The radiation detection capability was observed to be relatively higher, when the polarization of the input laser and the antenna is same.

  17. Laser induced plasma on copper target, a non-equilibrium model

    SciTech Connect

    Oumeziane, Amina Ait Liani, Bachir; Parisse, Jean-Denis

    2014-02-15

    The aim of this work is to present a comprehensive numerical model for the UV laser ablation of metal targets, it focuses mainly on the prediction of laser induced plasma thresholds, the effect of the laser-plasma interaction, and the importance of the electronic non-equilibrium in the laser induced plume and its expansion in the background gas. This paper describes a set of numerical models for laser-matter interaction between 193-248 and 355 nm lasers and a copper target. Along with the thermal effects inside the material resulting from the irradiation of the latter with the pulsed laser, the laser-evaporated matter interaction and the plasma formation are thoroughly modelled. In the laser induced plume, the electronic nonequilibrium and the laser beam absorption have been investigated. Our calculations of the plasmas ignition thresholds on copper targets have been validated and compared to experimental as well as theoretical results. Comparison with experiment data indicates that our results are in good agreement with those reported in the literature. Furthermore, the inclusion of electronic non-equilibrium in our work indicated that this important process must be included in models of laser ablation and plasma plume formation.

  18. Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting.

    PubMed

    Brink, Maartje C; van der Velden, Yme; de Leeuw, Wim; Mateos-Langerak, Julio; Belmont, Andrew S; van Driel, Roel; Verschure, Pernette J

    2006-01-01

    Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1alpha or HP1beta we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1alpha and HP1beta to the chromosome region. We show that CD-less HP1alpha can induce chromatin condensation, whereas the effect of truncated HP1beta is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization. PMID:16283356

  19. N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli

    PubMed Central

    Sekar, Karthik; Gentile, Andrew M.; Bostick, John W.; Tyo, Keith E. J.

    2016-01-01

    Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits. PMID:26900850

  20. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells

    PubMed Central

    Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-01-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  1. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells.

    PubMed

    Zou, Peng; Xia, Yiqun; Chen, Weiqian; Chen, Xi; Ying, Shilong; Feng, Zhiguo; Chen, Tongke; Ye, Qingqing; Wang, Zhe; Qiu, Chenyu; Yang, Shulin; Liang, Guang

    2016-04-01

    Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer effïcacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy. PMID:26919110

  2. Study on Wangzaozin-A-Inducing Cancer Apoptosis and Its Theoretical Protein Targets.

    PubMed

    Chen, Jing; Wang, Shixia; Lu, Xiaoquan

    2016-08-01

    Wangzaozin A is the most representative cytotoxic C-20-nonoxide compound of Isodon plants of Labiatae. The protein targets of the 2 isomers (X and X') of Wangzaozin A are, respectively, extracted from target fishing dock Web site. Each isomer has 23 targets with good energy scores. The binding modes of each isomer and its targets are, respectively, analyzed by Dock. The energy score of the binding mode between the isomer X and the inositol-1(or 4)-monophosphatase is the smallest. The apoptosis, antiproliferative, and lethal effects of human gastric cancer cells induced by Wangzaozin A are assessed by trypan blue exclusion, hoechst33258 stain in SGC-7901, and flow cytometric measurement. The mechanism of Wangzaozin-A-inducing cancer apoptosis is analyzed. Wangzaozin A inhibits the growth of human gastric cancer SGC-7901 cell lines at the lower concentration (<4.0 µmol/L) and results in the lethal effect at the relative higher concentration (>8.0 µmol/L). PMID:26082454

  3. Eupatilin inhibits EGF-induced JB6 cell transformation by targeting PI3K.

    PubMed

    Li, Feng; Tao, Ya; Qiao, Yan; Li, Ke; Jiang, Yanan; Cao, Chang; Ren, Shuxin; Chang, Xiaobin; Wang, Xiaona; Wang, Yanhong; Xie, Yifei; Dong, Ziming; Zhao, Jimin; Liu, Kangdong

    2016-09-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that play fundamental roles in regulation of multiple signaling pathways, including cell proliferation, survival and cell cycle. Increasing evidence has shown that abnormal activation of PI3K pathway contributes to tumorigenesis and progression of various malignant tumors. Therefore, it is an attractive target of chemoprevention and chemotherapy. Eupatilin, a natural flavone compound extracted from Artemisia vulgaris, has antitumor and anti-inflammation efficacy. However, the direct target(s) of eupatilin in cancer chemoprevention are still elusive. In the present study, we reported eupatilin suppressed JB6 cell proliferation and its EGF-induced colony formation. Eupatilin attenuated phosphorylation of PI3K downstream signaling molecules. Downregulation of cyclin D1 expression and arresting in G1 phase were induced through eupatilin treatment. Furthermore, we found it could bind to the p110α, a catalytic subunit of PI3K, by computational docking methods. Pull down assay outcomes also verified the binding of eupatilin with PI3K. Taken together, our results suggest that epatilin is a potential chemopreventive agent in inhibition of skin cell transformation by targeting PI3K. PMID:27573489

  4. Novel application of brain-targeting polyphenol compounds in sleep deprivation-induced cognitive dysfunction

    PubMed Central

    Zhao, Wei; Wang, Jun; Bi, Weina; Ferruzzi, Mario; Yemul, Shrishailam; Freire, Daniel; Mazzola, Paolo; Ho, Lap; Dubner, Lauren; Pasinetti, Giulio Maria

    2016-01-01

    Sleep deprivation produces deficits in hippocampal synaptic plasticity and hippocampal-dependent memory storage. Recent evidence suggests that sleep deprivation disrupts memory consolidation through multiple mechanisms, including the down-regulation of the cAMP-response element-binding protein (CREB) and of mammalian target of rapamycin (mTOR) signaling. In this study, we tested the effects of a Bioactive Dietary Polyphenol Preparation (BDPP), comprised of grape seed polyphenol extract, Concord grape juice, and resveratrol, on the attenuation of sleep deprivation-induced cognitive impairment. We found that BDPP significantly improves sleep deprivation-induced contextual memory deficits, possibly through the activation of CREB and mTOR signaling pathways. We also identified brain-available polyphenol metabolites from BDPP, among which quercetin-3-O-glucuronide activates CREB signaling and malvidin-3-O-glucoside activates mTOR signaling. In combination, quercetin and malvidin-glucoside significantly attenuated sleep deprivation-induced cognitive impairment in -a mouse model of acute sleep deprivation. Our data suggests the feasibility of using select brain-targeting polyphenol compounds derived from BDPP as potential therapeutic agents in promoting resilience against sleep deprivation-induced cognitive dysfunction. PMID:26235983

  5. Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

    PubMed

    Ma, Xiao-Hong; Piao, Sheng-Fu; Dey, Souvik; McAfee, Quentin; Karakousis, Giorgos; Villanueva, Jessie; Hart, Lori S; Levi, Samuel; Hu, Janice; Zhang, Gao; Lazova, Rossitza; Klump, Vincent; Pawelek, John M; Xu, Xiaowei; Xu, Wei; Schuchter, Lynn M; Davies, Michael A; Herlyn, Meenhard; Winkler, Jeffrey; Koumenis, Constantinos; Amaravadi, Ravi K

    2014-03-01

    Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway. PMID:24569374

  6. MiR-21 Protected Cardiomyocytes against Doxorubicin-Induced Apoptosis by Targeting BTG2

    PubMed Central

    Tong, Zhongyi; Jiang, Bimei; Wu, Yanyang; Liu, Yanjuan; Li, Yuanbin; Gao, Min; Jiang, Yu; Lv, Qinglan; Xiao, Xianzhong

    2015-01-01

    Doxorubicin (DOX) is an anthracycline drug with a wide spectrum of antineoplastic activities. However, it causes cardiac cytotoxicity, and this limits its clinical applications. MicroRNA-21 (miR-21) plays a vital role in regulating cell proliferation and apoptosis. While miR-21 is preferentially expressed in adult cardiomyocytes and involved in cardiac development and heart disease, little is known regarding its biological functions in responding to DOX-induced cardiac cytotoxicity. In this study, the effects of DOX on mouse cardiac function and the expression of miR-21 were examined in both mouse heart tissues and rat H9C2 cardiomyocytes. The results showed that the cardiac functions were more aggravated in chronic DOX injury mice compared with acute DOX-injury mice; DOX treatment significantly increased miR-21 expression in both mouse heart tissue and H9C2 cells. Over-expression of miR-21 attenuated DOX-induced apoptosis in cardiamyocytes whereas knocking down its expression increased DOX-induced apoptosis. These gain- and loss- of function experiments showed that B cell translocation gene 2 (BTG2) was a target of miR-21. The expression of BTG2 was significantly decreased both in myocardium and H9C2 cells treated with DOX. The present study has revealed that miR-21 protects mouse myocardium and H9C2 cells against DOX-induced cardiotoxicity probably by targeting BTG2. PMID:26132560

  7. Novel application of brain-targeting polyphenol compounds in sleep deprivation-induced cognitive dysfunction.

    PubMed

    Zhao, Wei; Wang, Jun; Bi, Weina; Ferruzzi, Mario; Yemul, Shrishailam; Freire, Daniel; Mazzola, Paolo; Ho, Lap; Dubner, Lauren; Pasinetti, Giulio Maria

    2015-10-01

    Sleep deprivation produces deficits in hippocampal synaptic plasticity and hippocampal-dependent memory storage. Recent evidence suggests that sleep deprivation disrupts memory consolidation through multiple mechanisms, including the down-regulation of the cAMP-response element-binding protein (CREB) and of mammalian target of rapamycin (mTOR) signaling. In this study, we tested the effects of a Bioactive Dietary Polyphenol Preparation (BDPP), comprised of grape seed polyphenol extract, Concord grape juice, and resveratrol, on the attenuation of sleep deprivation-induced cognitive impairment. We found that BDPP significantly improves sleep deprivation-induced contextual memory deficits, possibly through the activation of CREB and mTOR signaling pathways. We also identified brain-available polyphenol metabolites from BDPP, among which quercetin-3-O-glucuronide activates CREB signaling and malvidin-3-O-glucoside activates mTOR signaling. In combination, quercetin and malvidin-glucoside significantly attenuated sleep deprivation-induced cognitive impairment in -a mouse model of acute sleep deprivation. Our data suggests the feasibility of using select brain-targeting polyphenol compounds derived from BDPP as potential therapeutic agents in promoting resilience against sleep deprivation-induced cognitive dysfunction. PMID:26235983

  8. miR-320a mediates doxorubicin-induced cardiotoxicity by targeting VEGF signal pathway

    PubMed Central

    Yin, Zhongwei; Zhao, Yanru; Li, Huaping; Yan, Mengwen; Zhou, Ling; Chen, Chen; Wang, Dao Wen

    2016-01-01

    Background Vascular homeostasis abnormalities may involve in doxorubicin induced cardiotoxicity. Methods Enhanced cardiac miR-320a expression, reduced cardiac microvessel density and impaired cardiac function were observed in mice treated by anthracycline doxorubicin. To further explore the role of miR-320a in doxorubicin induced cardiotoxicity, microRNA mimics/inhibitor in vitro and rAAV administration in vivo were employed in mice. Results Knockdown of miR-320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR-320a enhanced apoptosis in vitro, and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice. Furthermore, Western blot assays showed that VEGF-A was a potential target of miR-320a, which was verified by anti-Ago2 co-immunoprecipitation. Moreover, as same as miR-320a, siRNA against VEGF-A reinforced doxorubicin induced endothelial cells injury. Finally, the negative effects of miR-320a on vascular homeostasis and cardiac function were alleviated by VEGF-A re-expression in doxorubicin treated mice. Conclusion Our observations demonstrate that miR-320a play important roles in doxorubicin induced cardiotoxicity via vessel homeostasis in heart and thus, inhibition of miR-320a may be applied to the treatment of cardiac dysfunction induced by anthracycline. PMID:26837315

  9. Autophagy in Alcohol-Induced Multiorgan Injury: Mechanisms and Potential Therapeutic Targets

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Huang, Heqing

    2014-01-01

    Autophagy is a genetically programmed, evolutionarily conserved intracellular degradation pathway involved in the trafficking of long-lived proteins and cellular organelles to the lysosome for degradation to maintain cellular homeostasis. Alcohol consumption leads to injury in various tissues and organs including liver, pancreas, heart, brain, and muscle. Emerging evidence suggests that autophagy is involved in alcohol-induced tissue injury. Autophagy serves as a cellular protective mechanism against alcohol-induced tissue injury in most tissues but could be detrimental in heart and muscle. This review summarizes current knowledge about the role of autophagy in alcohol-induced injury in different tissues/organs and its potential molecular mechanisms as well as possible therapeutic targets based on modulation of autophagy. PMID:25140315

  10. Ultrasensitive detection of target analyte-induced aggregation of gold nanoparticles using laser-induced nanoparticle Rayleigh scattering.

    PubMed

    Lin, Jia-Hui; Tseng, Wei-Lung

    2015-01-01

    Detection of salt- and analyte-induced aggregation of gold nanoparticles (AuNPs) mostly relies on costly and bulky analytical instruments. To response this drawback, a portable, miniaturized, sensitive, and cost-effective detection technique is urgently required for rapid field detection and monitoring of target analyte via the use of AuNP-based sensor. This study combined a miniaturized spectrometer with a 532-nm laser to develop a laser-induced Rayleigh scattering technique, allowing the sensitive and selective detection of Rayleigh scattering from the aggregated AuNPs. Three AuNP-based sensing systems, including salt-, thiol- and metal ion-induced aggregation of the AuNPs, were performed to examine the sensitivity of laser-induced Rayleigh scattering technique. Salt-, thiol-, and metal ion-promoted NP aggregation were exemplified by the use of aptamer-adsorbed, fluorosurfactant-stabilized, and gallic acid-capped AuNPs for probing K(+), S-adenosylhomocysteine hydrolase-induced hydrolysis of S-adenosylhomocysteine, and Pb(2+), in sequence. Compared to the reported methods for monitoring the aggregated AuNPs, the proposed system provided distinct advantages of sensitivity. Laser-induced Rayleigh scattering technique was improved to be convenient, cheap, and portable by replacing a diode laser and a miniaturized spectrometer with a laser pointer and a smart-phone. Using this smart-phone-based detection platform, we can determine whether or not the Pb(2+) concentration exceed the maximum allowable level of Pb(2+) in drinking water. PMID:25476277

  11. Pion-induced production of the Zc(3900 ) off a nuclear target

    NASA Astrophysics Data System (ADS)

    Huang, Yin; He, Jun; Liu, Xiang; Zhang, Hong Fei; Xie, Ju Jun; Chen, Xu Rong

    2016-02-01

    We investigate the possibility to study the charmoniumlike state Zc(3900 ) through the pion-induced production off a nuclear target. By using a high-energy pion beam, the Zc(3900 ) can be produced off a proton or nucleus though the Primakoff effect. The production amplitude is calculated in an effective Lagrangian approach combined with the vector dominance model. The total cross sections of the p (π-,Zc-(3900 )) and p (π-,Zc-(3900 )→J /ψ π-) reactions are calculated, and their order of magnitude is about 0.1 and 0.01 nb, respectively, with an assumption of branch ratio 10% for the Zc(3900 ) decay in J /ψ π channel. If the proton target is replaced by a nuclear target, the production of the Zc(3900 ) enhances obviously. The predicted total cross sections for the A (π-,Zc-(3900 )) and A (π-,Zc-(3900 )→J /ψ π-) reactions with A =12C or 208Pb are on the order of magnitude of 100 and 10 nb, respectively, which is about one thousand times larger than the cross sections off a proton target. Based on these results, we suggest the experimental study of the Zc(3900 ) by using high-energy pion beams with a nuclear target at facilities such as COMPASS and J-PARC.

  12. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.

    PubMed

    Byrne, Susan M; Ortiz, Luis; Mali, Prashant; Aach, John; Church, George M

    2015-02-18

    Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. PMID:25414332

  13. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

    PubMed Central

    Riskin, Arieh; Mond, Yehudit

    2015-01-01

    Background Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture. Methods Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. Results GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation. PMID:26886772

  14. Rationally rewiring the connectivity of the XylR/Pu regulatory node of the m-xylene degradation pathway in Pseudomonas putida.

    PubMed

    de Las Heras, Aitor; Martínez-García, Esteban; Domingo-Sananes, Maria Rosa; Fraile, Sofia; de Lorenzo, Víctor

    2016-04-18

    The XylR/Pu regulatory node of the m-xylene biodegradation pathway of Pseudomonas putida mt-2 is one of the most intricate cases of processing internal and external cues into a single controlling element. Despite this complexity, the performance of the regulatory system is determined in vivo only by the occupation of Pu by m-xylene-activated XylR and σ(54)-RNAP. The stoichiometry between these three elements defines natural system boundaries that outline a specific functional space. This space can be expanded artificially following different strategies that involve either the increase of XylR or σ(54) or both elements at the same time (each using a different inducer). In this work we have designed a new regulatory architecture that drives the system to reach a maximum performance in response to one single input. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu node could be amended by simultaneously increasing σ(54) and XylR in response to only natural inducers. The exacerbation of Pu activity in vivo was tested in strains bearing synthetic transposons encoding xylR and rpoN (the σ(54) coding gene) controlled also by Pu, thereby generating a P. putida strain with the XylR/Pu output controlled by two intertwined feed forward loops (FFLs). The lack of a negative feedback loop in the expression node enables Pu activity to reach its physiological maximum in response to a single input. Only competition for cell resources might ultimately check the upper activity limit of such a rewired m-xylene sensing device. PMID:26961967

  15. Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension.

    PubMed

    Adesina, Sherry E; Kang, Bum-Yong; Bijli, Kaiser M; Ma, Jing; Cheng, Juan; Murphy, Tamara C; Michael Hart, C; Sutliff, Roy L

    2015-10-01

    Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O(2)(•-) (mtO(2)(•-)) and H(2)O(2) (mtH(2)O(2)) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72 h increased mtO(2)(•-) and mtH(2)O(2). To assess the contribution of mtO(2)(•-) and mtH(2)O(2) to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (Tg(hSOD2)) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H(2)O(2) (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, Tg(hSOD2) mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO(2)(•-) and mtH(2)O(2). Targeting mtH(2)O(2) attenuates PH pathogenesis, whereas targeting mtO(2)(•-) exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H(2)O(2) release. These studies suggest that targeted reductions in mtH(2)O(2) generation may be particularly effective in preventing hypoxia-induced PH. PMID:26073127

  16. Mitochondria Death/Survival Signaling Pathways in Cardiotoxicity Induced by Anthracyclines and Anticancer-Targeted Therapies

    PubMed Central

    Montaigne, David; Hurt, Christopher; Neviere, Remi

    2012-01-01

    Anthracyclines remain the cornerstone of treatment in many malignancies but these agents have a cumulative dose relationship with cardiotoxicity. Development of cardiomyopathy and congestive heart failure induced by anthracyclines are typically dose-dependent, irreversible, and cumulative. Although past studies of cardiotoxicity have focused on anthracyclines, more recently interest has turned to anticancer drugs that target many proteins kinases, such as tyrosine kinases. An attractive model to explain the mechanism of this cardiotoxicity could be myocyte loss through cell death pathways. Inhibition of mitochondrial transition permeability is a valuable tool to prevent doxorubicin-induced cardiotoxicity. In response to anthracycline treatment, activation of several protein kinases, neuregulin/ErbB2 signaling, and transcriptional factors modify mitochondrial functions that determine cell death or survival through the modulation of mitochondrial membrane permeability. Cellular response to anthracyclines is also modulated by a myriad of transcriptional factors that influence cell fate. Several novel targeted chemotherapeutic agents have been associated with a small but worrying risk of left ventricular dysfunction. Agents such as trastuzumab and tyrosine kinase inhibitors can lead to cardiotoxicity that is fundamentally different from that caused by anthracyclines, whereas biological effects converge to the mitochondria as a critical target. PMID:22482055

  17. Brainstem Reticulospinal Neurons are Targets for Corticotropin-Releasing Factor-Induced Locomotion in Roughskin Newts

    PubMed Central

    Hubbard, Catherine S.; Dolence, E. Kurt; Rose, James D.

    2009-01-01

    Stress-induced release or central administration of corticotropin-releasing factor (CRF) enhances locomotion in a wide range of vertebrates, including the roughskin newt, Taricha granulosa. Although CRF’s stimulatory actions on locomotor behavior are well established, the target neurons through which CRF exerts this effect remain unknown. To identify these target neurons, we utilized a fluorescent conjugate of CRF (CRF-TAMRA 1) to track this peptide’s internalization into reticulospinal and other neurons in the medullary reticular formation (MRF), a region critically involved in regulating locomotion. Epifluorescent and confocal microscopy revealed that CRF-TAMRA 1 was internalized by diverse MRF neurons, including reticulospinal neurons retrogradely labeled with Cascade Blue dextran. In addition, we immunohistochemically identified a distinct subset of serotonin-containing neurons, located throughout the medullary raphé, that also internalized the fluorescent CRF-TAMRA 1 conjugate. Chronic single-unit recordings obtained from microwire electrodes in behaving newts revealed that intracerebroventricular (icv) administration of CRF-TAMRA 1 increased medullary neuronal firing and that appearance of this firing was associated with, and strongly predictive of, episodes of CRF-induced locomotion. Furthermore, icv administered CRF-TAMRA 1 produced behavioral and neurophysiological effects identical to equimolar doses of unlabeled CRF. Collectively, these findings provide the first evidence that CRF directly targets reticulospinal and serotonergic neurons in the MRF and indicate that CRF may enhance locomotion via direct effects on the hindbrain, including the reticulospinal system. PMID:19968991

  18. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  19. Laser-induced disruption of systemically administered liposomes for targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Mackanos, Mark A.; Larabi, Malika; Shinde, Rajesh; Simanovskii, Dmitrii M.; Guccione, Samira; Contag, Christopher H.

    2009-07-01

    Liposomal formulations of drugs have been shown to enhance drug efficacy by prolonging circulation time, increasing local concentration and reducing off-target effects. Controlled release from these formulations would increase their utility, and hyperthermia has been explored as a stimulus for targeted delivery of encapsulated drugs. Use of lasers as a thermal source could provide improved control over the release of the drug from the liposomes with minimal collateral tissue damage. Appropriate methods for assessing local release after systemic delivery would aid in testing and development of better formulations. We use in vivo bioluminescence imaging to investigate the spatiotemporal distribution of luciferin, used as a model small molecule, and demonstrate laser-induced release from liposomes in animal models after systemic delivery. These liposomes were tested for luciferin release between 37 and 45 °C in PBS and serum using bioluminescence measurements. In vivo studies were performed on transgenic reporter mice that express luciferase constitutively throughout the body, thus providing a noninvasive readout for controlled release following systemic delivery. An Nd:YLF laser was used (527 nm) to heat tissues and induce rupture of the intravenously delivered liposomes in target tissues. These data demonstrate laser-mediated control of small molecule delivery using thermally sensitive liposomal formulations.

  20. Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging.

    PubMed

    Park, Gaeun; Baek, Sohee; Kim, Jong-Eun; Lim, Tae-gyu; Lee, Charles C; Yang, Hee; Kang, Young-Gyu; Park, Jun Seong; Augustin, Martin; Mrosek, Michael; Lee, Chang Yong; Dong, Zigang; Huber, Robert; Lee, Ki Won

    2015-12-01

    While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in preventing and improving UVB-associated skin aging. PMID:26341390

  1. Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase

    SciTech Connect

    Read, David J.; Li Yong; Chao, Moses V.; Cavanagh, John B.; Glynn, Paul

    2010-05-15

    Single doses of organophosphorus compounds (OP) which covalently inhibit neuropathy target esterase (NTE) can induce lower-limb paralysis and distal damage in long nerve axons. Clinical signs of neuropathy are evident 3 weeks post-OP dose in humans, cats and chickens. By contrast, clinical neuropathy in mice following acute dosing with OPs or any other toxic compound has never been reported. Moreover, dosing mice with ethyloctylphosphonofluoridate (EOPF) - an extremely potent NTE inhibitor - causes a different (subacute) neurotoxicity with brain oedema. These observations have raised the possibility that mice are intrinsically resistant to neuropathies induced by acute toxic insult, but may incur brain oedema, rather than distal axonal damage, when NTE is inactivated. Here we provide the first report that hind-limb dysfunction and extensive axonal damage can occur in mice 3 weeks after acute dosing with a toxic compound, bromophenylacetylurea. Three weeks after acutely dosing mice with neuropathic OPs no clinical signs were observed, but distal lesions were present in the longest spinal sensory axons. Similar lesions were evident in undosed nestin-cre:NTEfl/fl mice in which NTE had been genetically-deleted from neural tissue. The extent of OP-induced axonal damage in mice was related to the duration of NTE inactivation and, as reported in chickens, was promoted by post-dosing with phenylmethanesulfonylfluoride. However, phenyldipentylphosphinate, another promoting compound in chickens, itself induced in mice lesions different from the neuropathic OP type. Finally, EOPF induced subacute neurotoxicity with brain oedema in both wild-type and nestin-cre:NTEfl/fl mice indicating that the molecular target for this effect is not neural NTE.

  2. Coordinated induction of Nrf2 target genes protects against iron nitrilotriacetate (FeNTA)-induced nephrotoxicity

    SciTech Connect

    Tanaka, Yuji; Aleksunes, Lauren M. |; Goedken, Michael J.; Chen, Chuan; Reisman, Scott A.; Manautou, Jose E.; Klaassen, Curtis D.

    2008-09-15

    The iron chelate, ferric nitrilotriacetate (FeNTA), induces acute proximal tubular necrosis as a consequence of lipid peroxidation and oxidative tissue damage. Chronic exposure of FeNTA leads to a high incidence of renal adenocarcinomas in rodents. NF-E2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles, and regulates the basal and inducible expression of numerous detoxifying and antioxidant genes. To determine the roles of Nrf2 in regulating renal gene expression and protecting against oxidative stress-induced kidney damage, wild-type and Nrf2-null mice were administered FeNTA. Renal Nrf2 protein translocated to the nucleus at 6h after FeNTA treatment. FeNTA increased mRNA levels of Nrf2 target genes, including NQO1, GCLC, GSTpi1/2, Mrp1, 2, and 4 in kidneys from wild-type mice, but not Nrf2-null mice. Protein expression of NQO1, a prototypical Nrf2 target gene, was increased in wild-type mice, with no change in Nrf2-null mice. FeNTA produced more nephrotoxicity in Nrf2-null mice than wild-type mice as indicated by higher serum urea nitrogen and creatinine levels, as more urinary NAG, stronger 4-hydroxynonenal protein adduct staining, and more extensive proximal tubule damage. Furthermore, pretreatment with CDDO-Im, a potent small molecule Nrf2 activator, protected mice against FeNTA-induced renal toxicity. Collectively, these results suggest that activation of Nrf2 protects mouse kidneys from FeNTA-induced oxidative stress damage by coordinately up-regulating the expression of cytoprotective genes.

  3. Thiazole Antibiotics Target FoxM1 and Induce Apoptosis in Human Cancer Cells

    PubMed Central

    Bhat, Uppoor G.; Halasi, Marianna; Gartel, Andrei L.

    2009-01-01

    Forkhead box M1 (FoxM1) oncogenic transcription factor represents an attractive therapeutic target in the fight against cancer, because it is overexpressed in a majority of human tumors. Recently, using a cell-based assay system we identified thiazole antibiotic Siomycin A as an inhibitor of FoxM1 transcriptional activity. Here, we report that structurally similar thiazole antibiotic, thiostrepton also inhibits the transcriptional activity of FoxM1. Furthermore, we found that these thiopeptides did not inhibit the transcriptional activity of other members of the Forkhead family or some non-related transcription factors. Further experiments revealed that thiazole antibiotics also inhibit FoxM1 expression, but not the expression of other members of the Forkhead box family. In addition, we found that the thiazole antibiotics efficiently inhibited the growth and induced potent apoptosis in human cancer cell lines of different origin. Thiopeptide-induced apoptosis correlated with the suppression of FoxM1 expression, while overexpression of FoxM1 partially protected cancer cells from the thiazole antibiotic-mediated cell death. These data suggest that Siomycin A and thiostrepton may specifically target FoxM1 to induce apoptosis in cancer cells and FoxM1 inhibitors/thiazole antibiotics could be potentially developed as novel anticancer drugs against human neoplasia. PMID:19440351

  4. Mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes.

    PubMed

    He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

    2014-01-01

    Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

  5. Preventing diet-induced obesity in mice by adipose tissue transformation and angiogenesis using targeted nanoparticles.

    PubMed

    Xue, Yuan; Xu, Xiaoyang; Zhang, Xue-Qing; Farokhzad, Omid C; Langer, Robert

    2016-05-17

    The incidence of obesity, which is recognized by the American Medical Association as a disease, has nearly doubled since 1980, and obesity-related comorbidities have become a major threat to human health. Given that adipose tissue expansion and transformation require active growth of new blood vasculature, angiogenesis offers a potential target for the treatment of obesity-associated disorders. Here we construct two peptide-functionalized nanoparticle (NP) platforms to deliver either Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) activator rosiglitazone (Rosi) or prostaglandin E2 analog (16,16-dimethyl PGE2) to adipose tissue vasculature. These NPs were engineered through self-assembly of a biodegradable triblock polymer composed of end-to-end linkages between poly(lactic-coglycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) and an endothelial-targeted peptide. In this system, released Rosi promotes both transformation of white adipose tissue (WAT) into brown-like adipose tissue and angiogenesis, which facilitates the homing of targeted NPs to adipose angiogenic vessels, thereby amplifying their delivery. We show that i.v. administration of these NPs can target WAT vasculature, stimulate the angiogenesis that is required for the transformation of adipose tissue, and transform WAT into brown-like adipose tissue, by the up-regulation of angiogenesis and brown adipose tissue markers. In a diet-induced obese mouse model, these angiogenesis-targeted NPs have inhibited body weight gain and modulated several serological markers including cholesterol, triglyceride, and insulin, compared with the control group. These findings suggest that angiogenesis-targeting moieties with angiogenic stimulator-loaded NPs could be incorporated into effective therapeutic regimens for clinical treatment of obesity and other metabolic diseases. PMID:27140638

  6. Aptamer/target binding-induced triple helix forming for signal-on electrochemical biosensing.

    PubMed

    Mao, Yinfei; Liu, Jinquan; He, Dinggen; He, Xiaoxiao; Wang, Kemin; Shi, Hui; Wen, Li

    2015-10-01

    Owing to its diversified structures, high affinity, and specificity for binding a wide range of non-nucleic acid targets, aptamer is a useful molecular recognition tool for the design of various biosensors. Herein, we report a new signal-on electrochemical biosensing platform which is based on an aptamer/target binding-induced strand displacement and triple-helix forming. The biosensing platform is composed of a signal transduction probe (STP) modified with a methylene blue (MB) and a sulfhydryl group, a triplex-forming oligonucleotides probe (TFO) and a target specific aptamer probe (Apt). Through hybridization with the TFO probe and the Apt probe, the self-assembled STP on Au electrode via Au-S bonding keeps its rigid structure. The MB on the STP is distal to the Au electrode surface. It is eT off state. Target binding releases the Apt probe and liberates the end of the MB tagged STP to fold back and form a triplex-helix structure with TFO (STP/TFO/STP), allowing MB to approach the Au electrode surface and generating measurable electrochemical signals (eT ON). As test for the feasibility and universality of this signal-on electrochemical biosensing platform, two aptamers which bind to adenosine triphosphate (ATP) and human α-thrombin (Tmb), respectively, are selected as models. The detection limit of ATP was 7.2 nM, whereas the detection limit of Tmb was 0.86 nM. PMID:26078174

  7. Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

    PubMed

    Oláh, Enikő; Pesti, Réka; Taller, Dénes; Havelda, Zoltán; Várallyay, Éva

    2016-09-01

    Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments. PMID:27283101

  8. Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

    PubMed

    Raynal, Noël J-M; Lee, Justin T; Wang, Youjun; Beaudry, Annie; Madireddi, Priyanka; Garriga, Judith; Malouf, Gabriel G; Dumont, Sarah; Dettman, Elisha J; Gharibyan, Vazganush; Ahmed, Saira; Chung, Woonbok; Childers, Wayne E; Abou-Gharbia, Magid; Henry, Ryan A; Andrews, Andrew J; Jelinek, Jaroslav; Cui, Ying; Baylin, Stephen B; Gill, Donald L; Issa, Jean-Pierre J

    2016-03-15

    Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer. Cancer Res; 76(6); 1494-505. ©2015 AACR. PMID:26719529

  9. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice.

    PubMed

    Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M; Gorodnya, Olena M; Ruchko, Mykhaylo V; Wilson, Glenn L; Gillespie, Mark N; Parker, James C

    2014-01-01

    The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA) damage and ventilator induced lung injury (VILI). In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII) which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP) was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH) and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group. PMID:25153040

  10. Near infrared laser-induced targeted cancer therapy using thermoresponsive polymer encapsulated gold nanorods.

    PubMed

    Zhang, Zhenjiang; Wang, Jing; Nie, Xin; Wen, Tao; Ji, Yinglu; Wu, Xiaochun; Zhao, Yuliang; Chen, Chunying

    2014-05-21

    External stimuli, such as ultrasound, magnetic field, and light, can be applied to activate in vivo tumor targeting. Herein, we fabricated polymer encapsulated gold nanorods to couple the photothermal properties of gold nanorods and the thermo- and pH-responsive properties of polymers in a single nanocomposite. The activation mechamism was thus transformed from heat to near-infrared (NIR) laser, which can be more easily controlled. Doxorubicin, a clinical anticancer drug, can be loaded into the nanocomposite through electrostatic interactions with high loading content up to 24%. The nanocomposite's accumulation in tumor post systematic administration can be significantly enhanced by NIR laser irradiation, providing a prerequisite for their therapeutic application which almost completely inhibited tumor growth and lung metastasis. Since laser can be manipulated very precisely and flexibly, the nanocomposite provides an ideally versatile platform to simultaneously deliver heat and anticancer drugs in a laser-activation mechanism with facile control of the area, time, and dosage. The NIR laser-induced targeted cancer thermo-chemotherapy without using targeting ligands represents a novel targeted anticancer strategy with facile control and practical efficacy. PMID:24773323

  11. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells.

    PubMed

    López-Quesada, C; Fontaine, A-S; Farré, A; Joseph, M; Selva, J; Egea, G; Ludevid, M D; Martín-Badosa, E; Montes-Usategui, M

    2014-07-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  12. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

    PubMed Central

    López-Quesada, C.; Fontaine, A.-S.; Farré, A.; Joseph, M.; Selva, J.; Egea, G.; Ludevid, M. D.; Martín-Badosa, E.; Montes-Usategui, M.

    2014-01-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  13. Isomer production in intermediate-energy deuteron-induced reactions on a gold target

    NASA Astrophysics Data System (ADS)

    Balabekyan, A. R.; Karapetyan, G. S.; Demekhina, N. A.; Drnoyan, D. R.; Zhemenik, V. I.; Adam, J.; Zavorka, L.; Solnyshkin, A. A.; Tsoupko-Sitnikov, V. M.; Guimarães, V.; Deppman, A.

    2016-05-01

    Residual nuclei formed at ground and isomeric states from the interaction of 4.4 GeV deuteron with a gold target have been measured and investigated by the induced-activity method. Eight isomeric and ground-state pairs of target residues in the mass range of 44

  14. Modeling and production of 240Am by deuteron-induced activation of a 240Pu target

    NASA Astrophysics Data System (ADS)

    Finn, Erin C.; McNamara, Bruce; Greenwood, Larry; Wittman, Richard; Soderquist, Charles; Woods, Vincent; VanDevender, Brent; Metz, Lori; Friese, Judah

    2015-04-01

    A novel reaction pathway for production of 240Am is reported. Models of reaction cross-sections in EMPIRE II suggest that deuteron-induced activation of a 240Pu target produces maximum yields of 240Am from 11.5 MeV incident deuterons. This activation had not been previously reported in the literature. A 240Pu target was activated under the modeled optimum conditions to produce 240Am. The modeled cross-section for the 240Pu(d, 2n)240Am reaction is on the order of 20-30 mbarn, but the experimentally estimated value is 5.6 ± 0.2 mbarn. We discuss reasons for the discrepancy as well as production of other Am isotopes that contaminate the final product.

  15. Modeling and production of 240Am by deuteron-induced activation of a 240Pu target

    SciTech Connect

    Finn, Erin C.; McNamara, Bruce K.; Greenwood, Lawrence R.; Wittman, Richard S.; Soderquist, Chuck Z.; Woods, Vincent T.; VanDevender, Brent A.; Metz, Lori A.; Friese, Judah I.

    2015-02-01

    A novel reaction pathway for production of 240Am is reported. Models of reaction cross-sections in EMPIRE II suggests that deuteron-induced activation of a 240Pu target produces maximum yields of 240Am from 11.5 MeV incident deuterons. This activation had not been previously reported in the literature. A 240Pu target was activated under the modeled optimum conditions to produce 240Am. The modeled cross-section for the 240Pu(d, 2n)240Am reaction is on the order of 20-30 mbarn, but the experimentally estimated value is 5.3 ± 0.2 mbarn. We discuss reasons for the discrepancy as well as production of other Am isotopes that contaminate the final product.

  16. Targeting One Carbon Metabolism with an Antimetabolite Disrupts Pyrimidine Homeostasis and Induces Nucleotide Overflow.

    PubMed

    Ser, Zheng; Gao, Xia; Johnson, Christelle; Mehrmohamadi, Mahya; Liu, Xiaojing; Li, Siqi; Locasale, Jason W

    2016-06-14

    Antimetabolites that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used antimetabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU reveals intracellular uracil accumulation, whereas deoxyuridine levels exhibited increased flux into the extracellular space, resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflow metabolism. Together, these findings identify a determinant of an antimetabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism. PMID:27264180

  17. The complex interactions between radiation induced non-targeted effects and cancer.

    PubMed

    Campa, Alessandro; Balduzzi, Maria; Dini, Valentina; Esposito, Giuseppe; Tabocchini, Maria Antonella

    2015-01-01

    Radiation induced non-targeted effects have been widely investigated in the last two decades for their potential impact on low dose radiation risk. In this paper we will give an overview of the most relevant aspects related to these effects, starting from the definition of the low dose scenarios. We will underline the role of radiation quality, both in terms of mechanisms of interaction with the biological matter and for the importance of charged particles as powerful tools for low dose effects investigation. We will focus on cell communication, representing a common feature of non-targeted effects, giving also an overview of cancer models that have explicitly considered such effects. PMID:24139968

  18. Targeting by AutophaGy proteins (TAG): Targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy

    PubMed Central

    Park, Sungwoo; Choi, Jayoung; Biering, Scott B.; Dominici, Erin; Williams, Lelia E.; Hwang, Seungmin

    2016-01-01

    ABSTRACT LC3 has been used as a marker to locate autophagosomes. However, it is also well established that LC3 can localize on various membranous structures other than autophagosomes. We recently demonstrated that the LC3 conjugation system (ATG7, ATG3, and ATG12–ATG5-ATG16L1) is required to target LC3 and IFNG (interferon, gamma)-inducible GTPases to the parasitophorus vacuole membrane (PVM) of a protist parasite Toxoplasma gondii and consequently for IFNG to control T. gondii infection. Here we show that not only LC3, but also its homologs (GABARAP, GABARAPL1, and GABARAPL2) localize on the PVM of T. gondii in a conjugation-dependent manner. Knockout/knockdown of all LC3 homologs led to a significant reduction in targeting of the IFNG-inducible GTPases to the PVM of T. gondii and the IFNG-mediated control of T. gondii infection. Furthermore, when we relocated the ATG12–ATG5-ATG16L1 complex, which specifies the conjugation site of LC3 homologs, to alternative target membranes, the IFNG-inducible GTPases were targeted to the new target membranes rather than the PVM of T. gondii. These data suggest that the localization of LC3 homologs onto a membrane by the LC3 conjugation system is necessary and sufficient for targeting of the IFNG-inducible GTPases to the membrane, implying Targeting by AutophaGy proteins (TAG). Our data further suggest that the conjugation of ubiquitin-like LC3 homologs to the phospholipids of membranes may change the destiny of the membranes beyond degradation through lysosomal fusion, as the conjugation of ubiquitin to proteins changes the destiny of the proteins beyond proteasomal degradation. PMID:27172324

  19. Development of CNS Active Target for Deuteron Induced Reactions with High Intensity Exotic Beam

    NASA Astrophysics Data System (ADS)

    Ota, Shinsuke; Tokieda, H.; Lee, C. S.; Kojima, R.; Watanabe, Y. N.; Corsi, A.; Dozono, M.; Gibelin, J.; Hashimoto, T.; Kawabata, T.; Kawase, S.; Kubono, S.; Kubota, Y.; Maeda, Y.; Matsubara, H.; Matsuda, Y.; Michimasa, S.; Nakao, T.; Nishi, T.; Obertelli, A.; Otsu, H.; Santamaria, C.; Sasano, M.; Takaki, M.; Tanaka, Y.; Leung, T.; Uesaka, T.; Yako, K.; Yamaguchi, H.; Zenihiro, J.; Takada, E.

    An active target system called CAT, has been developed aiming at the measurement of deuteron induced reactions with high intensity beams in inverse kinematics. The CAT consists of a time projection chamber using THGEM and an array of Si detectors or NaI scintilators. The effective gain for the recoil particle is deisgned to be 5 - 10 × 103, while one for the beam is reduced by 102 using mesh grid to match the amplified signal to the dynamic range same as the one for recoil particle. The structure of CAT and the effect of the mesh grid are reported.

  20. Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

    PubMed

    Iwasaki, Arihiro; Ohno, Osamu; Katsuyama, Shun; Morita, Maho; Sasazawa, Yukiko; Dan, Shingo; Simizu, Siro; Yamori, Takao; Suenaga, Kiyotake

    2015-11-15

    In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts. PMID:26428873

  1. Molecular targets in radiation-induced blood-brain barrier disruption

    SciTech Connect

    Nordal, Robert A.; Wong, C. Shun . E-mail: shun.wang@sw.ca

    2005-05-01

    Disruption of the blood-brain barrier (BBB) is a key feature of radiation injury to the central nervous system. Studies suggest that endothelial cell apoptosis, gene expression changes, and alteration of the microenvironment are important in initiation and progression of injury. Although substantial effort has been directed at understanding the impact of radiation on endothelial cells and oligodendrocytes, growing evidence suggests that other cell types, including astrocytes, are important in responses that include induced gene expression and microenvironmental changes. Endothelial apoptosis is important in early BBB disruption. Hypoxia and oxidative stress in the later period that precedes tissue damage might lead to astrocytic responses that impact cell survival and cell interactions. Cell death, gene expression changes, and a toxic microenvironment can be viewed as interacting elements in a model of radiation-induced disruption of the BBB. These processes implicate particular genes and proteins as targets in potential strategies for neuroprotection.

  2. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    PubMed

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients. PMID:24100054

  3. Fast and sensitive detection of indels induced by precise gene targeting.

    PubMed

    Yang, Zhang; Steentoft, Catharina; Hauge, Camilla; Hansen, Lars; Thomsen, Allan Lind; Niola, Francesco; Vester-Christensen, Malene B; Frödin, Morten; Clausen, Henrik; Wandall, Hans H; Bennett, Eric P

    2015-05-19

    The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis. PMID:25753669

  4. Targeting the tumor necrosis factor-related apoptosis-inducing ligand path in neuroblastoma.

    PubMed

    Yang, Xuezhong; Thiele, Carol J

    2003-07-18

    The identification of the tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) a few years ago generated considerable enthusiasm for it as a potential cancer therapeutic agent. This is because TRAIL shows potent apoptosis inducing activity in a wide spectrum of transformed cell lines but not in cell lines derived from normal tissue origin. As the details in the signal transduction pathway of TRAIL-induced apoptosis are clarified, various defects of TRAIL pathway have been identified in TRAIL resistant cancer cells. Neuroblastoma is the most common extracranial solid tumor in children and those with a poor prognosis require more sensitive therapies. Unlike other cancer cells, most neuroblastoma cell lines are resistant to TRAIL induced apoptosis and the resistance correlates with caspase 8 deficiency, which is attributed to the methylation of the gene. Interferon (IFN)-gamma induces caspase 8 expression in most neuroblastoma cell lines regardless of the methylation status but fails to sensitize most NB to TRAIL. Further analysis indicates a TRAIL receptor deficiency contributes to TRAIL resistance in NB. Multiple lesions suggest that this path may play an important role in tumorigenesis and/ or evasion from therapies. Furthermore it indicates that the clinical application of TRAIL in NB will require a multi-modality approach. Important questions remain unanswered: How does IFN-gamma induce caspase 8 and why is the induction heterogeneous? How to stimulate the caspase 8 induction in cells that fail to respond to IFN-gamma? How to target other TRAIL pathway lesions with the clinically feasible approaches? PMID:12880973

  5. Initial observations of cavitation-induced erosion of liquid metal spallation target vessels at the Spallation Neutron Source

    NASA Astrophysics Data System (ADS)

    McClintock, D. A.; Riemer, B. W.; Ferguson, P. D.; Carroll, A. J.; Dayton, M. J.

    2012-12-01

    During operation of the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory the mechanical properties of the AISI 316L target module are altered by high-energy neutron and proton radiation. The interior surfaces of the target vessel are also damaged by cavitation-induced erosion, which results from repetitive rapid heating of the liquid mercury by high-energy proton beam pulses. Until recently no observations of cavitation-induced erosion were possible for conditions fully prototypic to the SNS. Post-Irradiation Examination (PIE) of the first and second operational SNS targets was performed to gain insight into the radiation-induced changes in mechanical properties of the 316L target material and the extent of cavitation-induced erosion to the mercury vessel inner surfaces. Observations of cavitation-induced erosion of the first and second operational SNS target modules are presented here, including images of the target vessel interiors and specimens removed from the target beam-entrance regions.

  6. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    SciTech Connect

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, Andre; Gnanasekar, Munirathinam

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. Black-Right-Pointing-Pointer Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. Black-Right-Pointing-Pointer Knock down of RAGE abrogates prostate tumor growth in vivo. Black-Right-Pointing-Pointer Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  7. CD16 polymorphisms and NK activation induced by monoclonal antibody-coated target cells.

    PubMed

    Bowles, Julie A; Weiner, George J

    2005-09-01

    CD16 and natural killer (NK) cells appear to play a central role in mediating the anti-tumor effects of monoclonal antibody (mAb) therapy, yet little is known about changes in NK cells that result from interaction of the NK cells with mAb-coated tumor cells under physiologic conditions. We developed a system using peripheral blood mononuclear cells (PBMCs) and either transformed B cells or breast cancer cells to assess how mAbs impact on NK cell phenotype. Rituximab, apolizumab and trastuzumab induced modulation of CD16 and upregulation of CD54 on NK cells when the appropriate target cells were present. Higher concentrations of mAb were needed to induce these changes on NK cells from subjects with the lower affinity CD16 polymorphism. Phenotypic changes were greater in NK cells from subjects with the higher affinity polymorphism even when saturating concentrations of mAb were used, demonstrating increased concentration of mAb can overcome some, but not all, of the influence CD16 polymorphisms have on NK activation. These studies provide a straightforward and easily reproducible technique to measure the ability of mAb-coated tumor cells to activate NK cells in vitro which should be particularly useful as mAbs with varying affinity for both target antigen and Fc receptor (FcR) are developed. PMID:16109421

  8. Metabolic and hypoxic adaptation to anti-angiogenic therapy: a target for induced essentiality

    PubMed Central

    McIntyre, Alan; Harris, Adrian L

    2015-01-01

    Anti-angiogenic therapy has increased the progression-free survival of many cancer patients but has had little effect on overall survival, even in colon cancer (average 6–8 weeks) due to resistance. The current licensed targeted therapies all inhibit VEGF signalling (Table1). Many mechanisms of resistance to anti-VEGF therapy have been identified that enable cancers to bypass the angiogenic blockade. In addition, over the last decade, there has been increasing evidence for the role that the hypoxic and metabolic responses play in tumour adaptation to anti-angiogenic therapy. The hypoxic tumour response, through the transcription factor hypoxia-inducible factors (HIFs), induces major gene expression, metabolic and phenotypic changes, including increased invasion and metastasis. Pre-clinical studies combining anti-angiogenics with inhibitors of tumour hypoxic and metabolic adaptation have shown great promise, and combination clinical trials have been instigated. Understanding individual patient response and the response timing, given the opposing effects of vascular normalisation versus reduced perfusion seen with anti-angiogenics, provides a further hurdle in the paradigm of personalised therapeutic intervention. Additional approaches for targeting the hypoxic tumour microenvironment are being investigated in pre-clinical and clinical studies that have potential for producing synthetic lethality in combination with anti-angiogenic therapy as a future therapeutic strategy. PMID:25700172

  9. The {alpha}-induced thick-target {gamma}-ray yield from light elements

    SciTech Connect

    Heaton, R.K. |

    1994-10-01

    The {alpha}-induced thick-target {gamma}-ray yield from light elements has been measured in the energy range 5.6 MeV {le} E{sub {alpha}} {le} 10 MeV. The {gamma}-ray yield for > 2.1 MeV from thick targets of beryllium, boron nitride, sodium fluoride, magnesium, aluminum and silicon were measured using the {alpha}-particle beam from the Lawrence Berkeley Laboratories 88 in. cyclotron. The elemental yields from this experiment were used to construct the {alpha}-induced direct production {gamma}-ray spectrum from materials in the SNO detector, a large volume ultra-low background neutrino detector located in the Creighton mine near Sudbury, Canada. This background source was an order of magnitude lower than predicted by previous calculations. These measurements are in good agreement with theoretical calculations of this spectrum based on a statistical nuclear model of the reaction, with the gross high energy spectrum structure being reproduced to within a factor of two. Detailed comparison of experimental and theoretical excitation population distribution of several residual nuclei indicate the same level of agreement within experimental uncertainties.

  10. miR-155 suppresses bacterial clearance in Pseudomonas aeruginosa-induced keratitis by targeting Rheb.

    PubMed

    Yang, Kun; Wu, Minhao; Li, Meiyu; Li, Dandan; Peng, Anping; Nie, Xinxin; Sun, Mingxia; Wang, Jinli; Wu, Yongjian; Deng, Qiuchan; Zhu, Min; Chen, Kang; Yuan, Jin; Huang, Xi

    2014-07-01

    miR-155 (microRNA-155) is an important noncoding RNA in regulating host inflammatory responses. However, its regulatory role in ocular infection remains unclear. Our study first explored the function of miR-155 in Pseudomonas aeruginosa-induced keratitis, one of the most common sight-threatening ocular diseases. We found that miR-155 expression was enhanced in human and mouse corneas after P. aeruginosa infection and was mainly expressed in macrophages but not neutrophils. In vivo studies demonstrated that miR-155 knockout mice displayed more resistance to P. aeruginosa keratitis, with a higher inducible nitric oxide synthase level and a lower bacterial burden. More importantly, in vitro data indicated that miR-155 suppressed the macrophage-mediated bacterial phagocytosis and intracellular killing of P. aeruginosa by targeting Rheb (Ras homolog enriched in brain). To the best of our knowledge, this is the first study to explore the role of miR-155 in bacterial keratitis, which may provide a promising target for clinical treatment of P. aeruginosa keratitis and other infectious diseases. PMID:24403554

  11. Therapeutic targets for the management of peripheral nerve injury-induced neuropathic pain.

    PubMed

    Jaggi, Amteshwar Singh; Singh, Nirmal

    2011-08-01

    Neuropathic pain is a debilitating form of treatment resistant chronic pain and responds poorly to the clinically available therapies. Studies from animal models of neuropathic pain have led to understanding of its pathobiology which includes complex interrelated pathways leading to peripheral and central neuronal sensitization. Advancements in the elucidation of neuropathic pain mechanisms have revealed a number of key targets that have been hypothesized to modulate clinical status. The present review discusses these therapeutic targets including noradrenaline and 5-HT reuptake inhibitors; sodium, calcium and potassium channels; inhibitory and excitatory neurotransmitters; neuropeptides including bradykinin, tachykinin, cholecystokinin, neuropeptide Y, vasoactive intestinal peptide, and CGRP; pro-inflammatory cytokines; MAP kinases; PPAR γ; Na(+)/Ca(2+) exchanger; nitric oxide; purinergic receptors; neuronal nicotinic receptors; cation-dependent chloride transporters; oxidative stress; matrix metalloproteinase and plasminogen activators; growth factors; transient receptor potential (TRP) channels; endocannabinoids; histamine receptors; dopamine; sigma receptors, beta adrenergic receptors, endothelins, and D-amino acid oxidase. The exploitation of these targets may provide effective therapeutic agents for the management of peripheral nerve injury-induced neuropathic pain. PMID:21631400

  12. Recent results on fast electron production induced by energetic heavy ions on thin solid targets

    NASA Astrophysics Data System (ADS)

    Lanzanò, G.; Anzalone, A.; Arena, N.; De Filippo, E.; Geraci, M.; Giustolisi, F.; Pagano, A.; Rothard, H.; Volant, C.

    2003-08-01

    In order to study the emission of energetic electrons induced by the impact of swift heavy ions on thin solid targets, we carried out a series of experiments at the Superconducting Cyclotron of the Catania Laboratori Nazionali del Sud (LNS) in November and December 2001. We bombarded solid thin targets, ranging from carbon to bismuth, with different ion beams at fixed velocity, i.e. ˜23 MeV/nucleon 197Au 36+, 58Ni 14+ and 12C 3+. Absolute velocity spectra were measured in a wide laboratory angular range, from 1.5° to 175°. At forward angles, besides the well-known convoy and binary encounter components with the beam velocity and two times the beam velocity respectively, we observe also a high velocity tail and an intermediate velocity component. At backward laboratory angles, the spectra remain complex, still presenting an energetic tail. These electron velocity spectra strongly depend on the beam and target atomic numbers. We suggest a Fermi-Shuttle (or multiscattering) mechanism and an in-flight-emission of projectile Auger electrons to explain some of the observed features in the velocity spectra.

  13. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves.

    PubMed

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis. PMID:21950944

  14. MicroRNA-320 Induces Neurite Outgrowth by Targeting ARPP-1

    PubMed Central

    White, Robin E.; Giffard, Rona G.

    2012-01-01

    MicroRNAs are important in central nervous system development, functioning, and pathophysiology. Here we demonstrate that increasing levels of microRNA 320 (miR-320) for 3 days markedly increases neurite length and at 4 days reduces total cell number in N2A cells. In silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a (Sema3a) as potential targets that could be involved. ARPP-19 was validated by demonstrating reduced mRNA and protein levels when miR-320 was overexpressed, while miR-320 had no effect on Sema3a expression. ARPP-19 is known to inhibit protein phosphatase-2A (PP2A) activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus increased levels of miR-320 leads to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury. PMID:22617447

  15. miR-709 modulates LPS-induced inflammatory response through targeting GSK-3β.

    PubMed

    Li, Ming; Chen, Hu; Chen, Luxi; Chen, Yaosheng; Liu, Xiaohong; Mo, Delin

    2016-07-01

    MicroRNAs (miRNAs) are endogenous small non-coding RNAs which modulate gene expression at the post-transcriptional level by either translational inhibition or mRNA degradation. MicroRNAs play important roles in both innate and adaptive immune response, including TLR-triggered immune response. In this study, we found that the expression of miR-709 was up-regulated in primary macrophage and RAW264.7 cells during the stimulation of LPS. Overexpression of miR-709 in RAW264.7 cells led to reduced production and gene expression of inflammatory cytokines (IL-6, TNF-α, IL-1β) during activation by LPS, whereas knockdown of miR-709 had completely opposite effects. We used bioinformatics and experimental techniques to demonstrate that GSK-3β is a direct target of miR-709. miR-709 mimics decreased GSK-3β protein but not mRNA level. We also found that miR-709 regulated the LPS-induced inflammatory response by targeting GSK-3β and elevating β-catenin. In conclusion, our data revealed a novel role for miR-709 in regulation of inflammatory response by targeting GSK-3β. PMID:27232654

  16. Growth arrest specific protein 6 participates in DOCA-induced target-organ damage.

    PubMed

    Park, Joon-Keun; Theuer, Stefanie; Kirsch, Torsten; Lindschau, Carsten; Klinge, Uwe; Heuser, Arnd; Plehm, Ralph; Todiras, Mihai; Carmeliet, Peter; Haller, Hermann; Luft, Friedrich C; Muller, Dominik N; Fiebeler, Anette

    2009-08-01

    Growth arrest-specific protein 6 (Gas 6) is involved in inflammatory kidney diseases, vascular remodeling, cell adhesion, and thrombus formation. We explored a role for Gas 6 in aldosterone-induced target organ damage. We observed that Gas 6 was upregulated in rats with high aldosterone levels. Mineralocorticoid receptor blockade prevented target organ damage and decreased the elevated Gas 6 expression. Vascular smooth muscle cells given aldosterone increased their Gas 6 expression in vitro. To test the pathophysiological relevance, we investigated the effects of deoxycorticosterone acetate (DOCA) on Gas 6 gene-deleted ((-/-)) mice. After 6 weeks DOCA, Gas 6(-/-) mice developed similar telemetric blood pressure elevations compared to wild-type mice but were protected from cardiac hypertrophy. Cardiac expression of interleukin 6 and collagen IV was blunted in Gas 6(-/-) mice, indicating reduced inflammation and fibrosis. Gas 6(-/-) mice also had an improved renal function with reduced albuminuria, compared to wild-type mice. Renal fibrosis and fibronectin deposition in the kidney were also reduced. Gas 6 deficiency reduces the detrimental effects of aldosterone on cardiac and renal remodeling independent of blood pressure reduction. Gas 6 appears to play a role in mineralocorticoid receptor-mediated target organ damage. Furthermore, because warfarin interferes with Gas 6 protein expression, the findings could be of clinical relevance for anticoagulant choices. PMID:19564549

  17. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  18. IKK is a therapeutic target in KRAS-Induced lung cancer with disrupted p53 activity

    PubMed Central

    Bassères, Daniela S.; Ebbs, Aaron; Cogswell, Patricia C.; Baldwin, Albert S.

    2014-01-01

    Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-KrasG12D) combined with loss of p53 (LSL-KrasG12D/p53fl/fl). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity. PMID:24955217

  19. Targeting Renal Purinergic Signalling for the Treatment of Lithium-induced Nephrogenic Diabetes Insipidus

    PubMed Central

    Kishore, B. K.; Carlson, N. G.; Ecelbarger, C. M.; Kohan, D. E.; Müller, C. E.; Nelson, R. D.; Peti-Peterdi, J.; Zhang, Y.

    2015-01-01

    Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development nephrogenic diabetes insipidus (NDI), a debilitating condition. Lithium-induced NDI is due to resistance of the kidney to arginine vasopressin (AVP), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides (ATP/UTP), acting via P2Y receptors, opposes the action of AVP on renal collecting duct (CD) by decreasing the cellular cAMP and thus AQP2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP/UTP-activated P2Y2 receptor in lithium-induced NDI in rats, and showed that P2Y2 receptor knockout mice are significantly resistant to Li-induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP-activated P2Y12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulfate (Plavix®) ameliorates Li-induced NDI in rodents. Parallel in vitro studies showed that P2Y12 receptor blockade by the reversible antagonist PSB-0739 sensitizes CD to the action of AVP. Thus, our studies unraveled the potential beneficial effects of targeting P2Y2 or P2Y12 receptors to counter AVP resistance in lithium-induced NDI. If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti-AVP effects to those that enhance the sensitivity of the kidney to AVP action. PMID:25877068

  20. Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells.

    PubMed

    Jarvis, Reagan M; Göttert, Jana; Murphy, Michael P; Ledgerwood, Elizabeth C

    2007-09-01

    Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid-soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ(3), MitoQ(5), MitoQ(10) and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death. PMID:17729122

  1. Intramuscular Therapeutic Vaccination Targeting HPV16 Induces T Cell Responses That Localize in Mucosal Lesions

    PubMed Central

    Jotova, Iveta; Wu, T. C.; Wang, Chenguang; Desmarais, Cindy; Boyer, Jean D.; Tycko, Benjamin; Robins, Harlan S.; Clark, Rachael A.; Trimble, Cornelia L.

    2014-01-01

    About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8+ T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation. PMID:24477000

  2. Intramuscular therapeutic vaccination targeting HPV16 induces T cell responses that localize in mucosal lesions.

    PubMed

    Maldonado, Leonel; Teague, Jessica E; Morrow, Matthew P; Jotova, Iveta; Wu, T C; Wang, Chenguang; Desmarais, Cindy; Boyer, Jean D; Tycko, Benjamin; Robins, Harlan S; Clark, Rachael A; Trimble, Cornelia L

    2014-01-29

    About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8(+) T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation. PMID:24477000

  3. Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

    PubMed

    Ahmad, Shama; Ahmad, Aftab; Hendry-Hofer, Tara B; Loader, Joan E; Claycomb, William C; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L; Chandler, Joshua D; Day, Brian J; Veress, Livia A; White, Carl W

    2015-04-01

    Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881

  4. Targeting Pin1 Protects Mouse Cardiomyocytes from High-Dose Alcohol-Induced Apoptosis

    PubMed Central

    Wang, Yuehong; Li, Zizhuo; Zhang, Yu; Yang, Wei; Sun, Jiantao; Shan, Lina; Li, Weimin

    2016-01-01

    Long-term heavy alcohol consumption is considered to be one of the main causes of left ventricular dysfunction in alcoholic cardiomyopathy (ACM). As previously suggested, high-dose alcohol induces oxidation stress and apoptosis of cardiomyocytes. However, the underlying mechanisms are yet to be elucidated. In this study, we found that high-dose alcohol treatment stimulated expression and activity of Pin1 in mouse primary cardiomyocytes. While siRNA-mediated knockdown of Pin1 suppressed alcohol-induced mouse cardiomyocyte apoptosis, overexpression of Pin1 further upregulated the numbers of apoptotic mouse cardiomyocytes. We further demonstrated that Pin1 promotes mitochondria oxidative stress and loss of mitochondrial membrane potential but suppresses endothelial nitric oxide synthase (eNOS) expression in the presence of alcohol. Taken together, our results revealed a pivotal role of Pin1 in regulation of alcohol-induced mouse cardiomyocytes apoptosis by promoting reactive oxygen species (ROS) accumulation and repressing eNOS expression, which could be potential therapeutic targets for ACM. PMID:26697133

  5. Th17/Treg Imbalance Induced by Dietary Salt Variation Indicates Inflammation of Target Organs in Humans.

    PubMed

    Luo, Tao; Ji, Wen-Jie; Yuan, Fei; Guo, Zhao-Zeng; Li, Yun-Xiao; Dong, Yan; Ma, Yong-Qiang; Zhou, Xin; Li, Yu-Ming

    2016-01-01

    The functions of T helper 17 (Th17) and regulatory T (Treg) cells are tightly orchestrated through independent differentiation pathways that are involved in the secretion of pro- and anti-inflammatory cytokines induced by high-salt dietary. However, the role of imbalanced Th17/Treg ratio implicated in inflammation and target organ damage remains elusive. Here, by flow cytometry analysis, we demonstrated that switching to a high-salt diet resulted in decreased Th17 cells and reciprocally increased Treg cells, leading to a decreased Th17/Treg ratio. Meanwhile, Th17-related pathway was down-regulated after one day of high salt loading, with the increase in high salt loading as shown by microarray and RT-PCR. Subsequently, blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) observed hypoxia in the renal medulla (increased R2(*) signal) during high-salt loading, which was regressed to its baseline level in a step-down fashion during low-salt feeding. The flow-mediated vasodilatation (FMD) of the branchial artery was significantly higher on the first day of high salt loading. Collectively, these observations indicate that a short-term increase in dietary salt intake could induce reciprocal switches in Th17/Treg ratio and related cytokines, which might be the underlying cellular mechanism of high-salt dietary induced end organ inflammation and potential atherosclerotic risk. PMID:27353721

  6. Th17/Treg Imbalance Induced by Dietary Salt Variation Indicates Inflammation of Target Organs in Humans

    PubMed Central

    Luo, Tao; Ji, Wen-jie; Yuan, Fei; Guo, Zhao-zeng; Li, Yun-xiao; Dong, Yan; Ma, Yong-qiang; Zhou, Xin; Li, Yu-ming

    2016-01-01

    The functions of T helper 17 (Th17) and regulatory T (Treg) cells are tightly orchestrated through independent differentiation pathways that are involved in the secretion of pro- and anti-inflammatory cytokines induced by high-salt dietary. However, the role of imbalanced Th17/Treg ratio implicated in inflammation and target organ damage remains elusive. Here, by flow cytometry analysis, we demonstrated that switching to a high-salt diet resulted in decreased Th17 cells and reciprocally increased Treg cells, leading to a decreased Th17/Treg ratio. Meanwhile, Th17-related pathway was down-regulated after one day of high salt loading, with the increase in high salt loading as shown by microarray and RT-PCR. Subsequently, blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) observed hypoxia in the renal medulla (increased R2* signal) during high-salt loading, which was regressed to its baseline level in a step-down fashion during low-salt feeding. The flow-mediated vasodilatation (FMD) of the branchial artery was significantly higher on the first day of high salt loading. Collectively, these observations indicate that a short-term increase in dietary salt intake could induce reciprocal switches in Th17/Treg ratio and related cytokines, which might be the underlying cellular mechanism of high-salt dietary induced end organ inflammation and potential atherosclerotic risk. PMID:27353721

  7. Chronic inflammation in biomaterial induced periprosthetic osteolysis: NF-κB as a therapeutic target

    PubMed Central

    Lin, Tzu-hua; Tamaki, Yasunobu; Pajarinen, Jukka; Waters, Heather A.; Woo, Deanna K.; Yao, Zhenyu; Goodman, Stuart B.

    2013-01-01

    Biomaterial-induced tissue responses in patients with total joint replacement are associated with the generation of wear particles, which may lead to chronic inflammation and local bone destruction (periprosthetic osteolysis). Inflammatory reactions associated with wear particles are mediated by several important signaling pathways, the most important of which involves the transcription factor NF-κB. NF-κB activation is essential for macrophage recruitment and maturation, as well as the production of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, MCP1, etc. In addition, NF-κB activation contributes to osteoclast differentiation and maturation via RANK/RANKL signaling, which increases bone destruction and reduces bone formation. Targeting individual downstream cytokines directly (such as TNF-α or IL-1β) may not effectively prevent wear particle induced osteolysis. A more logical upstream therapeutic approach may be provided by direct modulation of the core IκB/IKKα/β/NF-κB signaling pathway in the local environment, however, the timing, dose, and strategy for administration should be considered. Suppression of chronic inflammation via inhibition of NF-κB activity in patients with malfunctioning joint replacements may be an effective strategy to mitigate wear particle induced periprosthetic osteolysis. PMID:24090989

  8. Peroxiredoxin-5 targeted to the mitochondrial intermembrane space attenuates hypoxia-induced reactive oxygen species signalling.

    PubMed

    Sabharwal, Simran S; Waypa, Gregory B; Marks, Jeremy D; Schumacker, Paul T

    2013-12-15

    The ability to adapt to acute and chronic hypoxia is critical for cellular survival. Two established functional responses to hypoxia include the regulation of gene transcription by HIF (hypoxia-inducible factor), and the constriction of pulmonary arteries in response to alveolar hypoxia. The mechanism of O2 sensing in these responses is not established, but some studies implicate hypoxia-induced mitochondrial ROS (reactive oxygen species) signalling. To further test this hypothesis, we expressed PRDX5 (peroxiredoxin-5), a H2O2 scavenger, in the IMS (mitochondrial intermembrane space), reasoning that the scavenging of ROS in that compartment should abrogate cellular responses triggered by the release of mitochondrial oxidants to the cytosol. Using adenoviral expression of IMS-PRDX5 (IMS-targeted PRDX5) in PASMCs (pulmonary artery smooth muscle cells) we show that IMS-PRDX5 inhibits hypoxia-induced oxidant signalling in the IMS and cytosol. It also inhibits HIF-1α stabilization and HIF activity in a dose-dependent manner without disrupting cellular oxygen consumption. IMS-PRDX5 expression also attenuates the increase in cytosolic [Ca(2+)] in PASMCs during hypoxia. These results extend previous work by demonstrating the importance of IMS-derived ROS signalling in both the HIF and lung vascular responses to hypoxia. PMID:24044889

  9. Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

    PubMed Central

    Ahmad, Aftab; Hendry-Hofer, Tara B.; Loader, Joan E.; Claycomb, William C.; Mozziconacci, Olivier; Schöneich, Christian; Reisdorph, Nichole; Powell, Roger L.; Chandler, Joshua D.; Day, Brian J.; Veress, Livia A.; White, Carl W.

    2015-01-01

    Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure. PMID:25188881

  10. Hot fusion-evaporation cross sections of 45Sc -induced reactions with lanthanide targets

    NASA Astrophysics Data System (ADS)

    Werke, T. A.; Mayorov, D. A.; Alfonso, M. C.; Bennett, M. E.; DeVanzo, M. J.; Frey, M. M.; Tereshatov, E. E.; Folden, C. M.

    2015-09-01

    Background: 45Sc has rarely been studied as a projectile in fusion-evaporation reactions. The synthesis of new superheavy elements with Z >118 will require projectiles with Z >20 , and 45Sc could potentially be used for this purpose. Purpose: Cross sections were measured for the x n and p x n exit channels in the reactions of 45Sc with lanthanide targets for comparison to previous measurements of 48Ca reacting with similar targets. These data provide insight on the survival of spherical, shell-stabilized nuclei against fission, and could have implications for the discovery of new superheavy elements. Methods: Beams of 45Sc6 + were delivered from the K500 superconducting cyclotron at Texas A&M University with an energy of ≈5 MeV /nucleon . Products were purified using the Momentum Achromat Recoil Spectrometer, and excitation functions were measured for reactions of 45Sc+156-158,160Gd, 159Tb , and 162Dy at five or more energies each. Evaporation residues were identified by their characteristic α -decay energies. Experimental data were compared to a simple theoretical model to study each step in the fusion-evaporation process. Results: The maximum measured 4 n cross sections for the reactions 45Sc+156-158,160Gd, 159Tb , and 162Dy are 5.8 ±1.7 , 25 ±5 , 39 ±7 , 150 ±20 , 2 .4-1.4+2.3 , and 1.8 ±0.6 μ b , respectively. Proton emission competes effectively with neutron emission from the excited compound nucleus in most cases. The α ,α n , and α 2 n products were also observed in the 45Sc+162Dy reaction. Conclusions: Excitation functions were reported for 45Sc -induced reactions on lanthanide targets for the first time, and these cross sections are much smaller than for 48Ca -induced reactions on the same targets. The relative neutron-deficiency of the compound nuclei leads to significantly increased fissility and large reductions in the survival probability. Little evidence for improved production cross sections due to shell-stabilization was observed.

  11. Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury.

    PubMed

    Filosto, Simone; Castillo, Sianna; Danielson, Aaron; Franzi, Lisa; Khan, Elaine; Kenyon, Nick; Last, Jerold; Pinkerton, Kent; Tuder, Rubin; Goldkorn, Tzipora

    2011-03-01

    Chronic obstructive pulmonary disease (COPD) is caused by exposure to cigarette smoke (CS). One mechanism of CS-induced lung injury is aberrant generation of ceramide, which leads to elevated apoptosis of epithelial and endothelial cells in the alveolar spaces. Recently, we discovered that CS-induced ceramide generation and apoptosis in pulmonary cells is governed by neutral sphingomyelinase (nSMase) 2. In the current experiments, we expanded our studies to investigate whether nSMase2 governs ceramide generation and apoptosis in vivo using rodent and human models of CS-induced lung injury. We found that exposure of mice or rats to CS leads to colocalizing elevations of ceramide levels and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling-positive cells in lung tissues. These increases are nSMase2 dependent, and are abrogated by treatment with N-acetyl cysteine or anti-nSMase2 small interfering RNA (siRNA). We further showed that mice that are heterozygous for nSMase2 demonstrate significant decrease in ceramide generation after CS exposure, whereas acidic sphingomyelinase (aSMase) knockout mice maintain wild-type ceramide levels, confirming our previous findings (in human airway epithelial cells) that only nSMase2, and not aSMase, is activated by CS exposure. Lastly, we found that lung tissues from patients with emphysema (smokers) display significantly higher levels of nSMase2 expression compared with lung tissues from healthy control subjects. Taken together, these data establish the central in vivo role of nSMase2 in ceramide generation, aberrant apoptosis, and lung injury under CS exposure, underscoring its promise as a novel target for the prevention of CS-induced airspace destruction. PMID:20448054

  12. Hypoxia-Inducible Factor (HIF) as a Target for Novel Therapies in Rheumatoid Arthritis

    PubMed Central

    Hua, Susan; Dias, Thilani H.

    2016-01-01

    Hypoxia is an important micro-environmental characteristic of rheumatoid arthritis (RA). Hypoxia-inducible factors (HIF) are key transcriptional factors that are highly expressed in RA synovium to regulate the adaptive responses to this hypoxic milieu. Accumulating evidence supports hypoxia and HIFs in regulating a number of important pathophysiological characteristics of RA, including synovial inflammation, angiogenesis, and cartilage destruction. Experimental and clinical data have confirmed the upregulation of both HIF-1α and HIF-2α in RA. This review will focus on the differential expression of HIFs within the synovial joint and its functional behavior in different cell types to regulate RA progression. Potential development of new therapeutic strategies targeting HIF-regulated pathways at sites of disease in RA will also be addressed. PMID:27445820

  13. Target-Induced and Equipment-Free DNA Amplification with a Simple Paper Device.

    PubMed

    Liu, Meng; Hui, Christy Y; Zhang, Qiang; Gu, Jimmy; Kannan, Balamurali; Jahanshahi-Anbuhi, Sana; Filipe, Carlos D M; Brennan, John D; Li, Yingfu

    2016-02-18

    We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps. PMID:26748431

  14. Smart bombing a single targeted cell with femtogram order reagents using laser-induced shockwave technique

    NASA Astrophysics Data System (ADS)

    Okano, Kazunori; Takizawa, Noriko; Uwada, Takayuki; Hosokawa, Yoichiroh; Masuhara, Hiroshi

    2008-02-01

    Injection and delivery of small amount reagent in aqueous solution for cell chip was performed utilizing regeneratively amplified femtosecond laser system. In our new trial, the reagent integrated on a solid strip are released and delivered to targeted cells with the femutosecond laser-induced impulsive-force. The reagent was fixed in poly(vinyl alcohol) or polystyrene film on a glass-substrate strip. When a single pulsed femtosecond laser was focused in the solution, the film near the focal point was fragmented and the reagent was dispersed in 45-μm φ area at 50 μm from the surface of the reagent strip. As examples cardiomyocyte beating cells of P19CL6 were bombed with epinephrine and acetylcholine, and as a result the beating ratio of the cells were quickly stimulated and suppressed, respectively. The results demonstrate that the present method is a promising key nano/micro technology for diagnosis and drug discovery.

  15. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation induced death

    PubMed Central

    Atkinson, Jeffrey; Kapralov, Alexandr A.; Yanamala, Naveena; Tyurina, Yulia Y.; Amoscato, Andrew A.; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K.; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A.; Tyurin, Vladimir A.; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I.; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A.; Bayîr, Hülya; Pitt, Bruce R.; Epperly, Michael W.; Greenberger, Joel S.; Kagan, Valerian E.

    2013-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation. PMID:21988913

  16. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

    PubMed

    Atkinson, Jeffrey; Kapralov, Alexandr A; Yanamala, Naveena; Tyurina, Yulia Y; Amoscato, Andrew A; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A; Tyurin, Vladimir A; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A; Bayîr, Hülya; Pitt, Bruce R; Epperly, Michael W; Greenberger, Joel S; Kagan, Valerian E

    2011-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation. PMID:21988913

  17. MicroRNA-29a induces insulin resistance by targeting PPARδ in skeletal muscle cells

    PubMed Central

    ZHOU, YUEHUA; GU, PINGQING; SHI, WEIJIE; LI, JINGYUN; HAO, QUN; CAO, XIAOMEI; LU, QIN; ZENG, YU

    2016-01-01

    Intrauterine growth retardation (IUGR) induces metabolic syndrome, which is often characterized by insulin resistance (IR), in adults. Previous research has shown that microRNAs (miRNAs or miRs) play a role in the target genes involved in this process, but the mechanisms remain unclear. In the present study, we examined miRNA profiles using samples of skeletal muscles from both IUGR and control rat offspring whose mothers were fed either a protein-restricted diet or a diet which involved normal amounts of protein during pregnancy, respectively. miR-29a was found to be upregulated in the skeletal muscles of IUGR offspring. The luciferase reporter assay confirmed the direct interaction between miR-29a and peroxisome proliferator-activated receptor δ (PPARδ). Overexpression of miR-29a in the skeletal muscle cell line C2C12 suppressed the expression of its target gene PPARδ, which, in turn, influenced the expression of its coactivator, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Thus, PPARδ/PGC-1α-dependent signals together reduced insulin-dependent glucose uptake and adenosine triphosphate (ATP) production. Overexpression of miR-29a also caused a decrease in levels of glucose transporter 4 (GLUT4), the most important glucose transporter in skeletal muscle, which partially induced a decrease insulin-dependent glucose uptake. These findings provide evidence for a novel micro-RNA-mediated mechanism of PPARδ regulation, and we also noted the IR-promoting actions of miR-29a in skeletal muscles of IUGR. PMID:26936652

  18. Periodontitis in Rats Induces Systemic Oxidative Stress That Is Controlled by Bone-Targeted Antiresorptives

    PubMed Central

    Oktay, Sehkar; Chukkapalli, Sasanka S.; Rivera-Kweh, Mercedes F.; Velsko, Irina M.; Holliday, L. Shannon; Kesavalu, Lakshmyya

    2015-01-01

    Background Periodontitis is a chronic, polymicrobial inflammatory disease that degrades connective tissue and alveolar bone and results in tooth loss. Oxidative stress has been linked to the onset of periodontal tissue breakdown and systemic inflammation, and the success of antiresorptive treatments will rely on how effectively they can ameliorate periodontal disease–induced oxidative stress during oral infection. Methods Rats were infected with polybacterial inoculum consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, as an oral lavage every other week for 12 weeks. Daily subcutaneous injections of enoxacin, bisenoxacin, alendronate, or doxycycline were administered for 6 weeks after 6 weeks of polybacterial infection in rats. The serum levels of oxidative stress parameters and antioxidant enzymes, including glutathione peroxidase, superoxide dismutase, and catalase, were evaluated in each of the infected, treated, and sham-infected rats. Results Rats infected with the periodontal pathogens displayed a five-fold increase in the oxidative stress index compared with controls as a result of increased levels of serum oxidants and decreases in total antioxidant activity. The overall decrease in antioxidant activity occurred despite increases in three important antioxidant enzymes, suggesting an imbalance between antioxidant macromolecules/small molecules production and antioxidant enzyme levels. Surprisingly, the bone-targeted antiresorptives bis-enoxacin and alendronate inhibited increases in oxidative stress caused by periodontitis. Bis-enoxacin, which has both antiresorptive and antibiotic activities, was more effective than alendronate, which acts only as an antiresorptive. Conclusion To the best of the authors’ knowledge, this is the first study to demonstrate that the increased oxidative stress induced by periodontal infection in rats can be ameliorated by bone-targeted antiresorptives. PMID:25101489

  19. Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium

    PubMed Central

    Wang, Hsiao-Lin V.; Dinwiddie, Brandon L.; Lee, Herman

    2015-01-01

    Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3′ UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr-siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. PMID:25480817

  20. Targeting hedgehog signalling by arsenic trioxide reduces cell growth and induces apoptosis in rhabdomyosarcoma.

    PubMed

    Boehme, Karen A; Zaborski, Julian J; Riester, Rosa; Schweiss, Sabrina K; Hopp, Ulrike; Traub, Frank; Kluba, Torsten; Handgretinger, Rupert; Schleicher, Sabine B

    2016-02-01

    Rhabdomyosarcomas (RMS) are soft tissue tumours treated with a combination of surgery and chemotherapy. However, mortality rates remain high in case of recurrences and metastatic disease due to drug resistance and failure to undergo apoptosis. Therefore, innovative approaches targeting specific signalling pathways are urgently needed. We analysed the impact of different hedgehog (Hh) pathway inhibitors on growth and survival of six RMS cell lines using MTS assay, colony formation assay, 3D spheroid cultures, flow cytometry and western blotting. Especially the glioma-associated oncogene family (GLI) inhibitor arsenic trioxide (ATO) effectively reduced viability as well as clonal growth and induced cell death in RMS cell lines of embryonal, alveolar and sclerosing, spindle cell subtype, whereas normal skeletal muscle cells were hardly compromised by ATO. Combination of ATO with itraconazole potentiated the reduction of colony formation and spheroid size. These results show that ATO is a promising substance for treatment of relapsed and refractory RMS by directly targeting GLI transcription factors. The combination with itraconazole or other chemotherapeutic drugs has the opportunity to enforce the treatment efficiency of resistant and recurrent RMS. PMID:26676886

  1. Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    PubMed Central

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  2. Blister-inducing antibodies target multiple epitopes on collagen VII in mice.

    PubMed

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-09-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  3. Relaxin Does Not Improve Angiotensin II-Induced Target-Organ Damage

    PubMed Central

    Haase, Nadine; Rugor, Julianna; Przybyl, Lukasz; Qadri, Fatimunnisa; Müller, Dominik N.; Dechend, Ralf

    2014-01-01

    Relaxin is a corpus-luteum produced protein hormone with vasodilatatory, anti-fibrotic, and angiogenic properties that are opposite to angiotensin (Ang) II. We investigated whether or not relaxin ameliorates Ang II-induced target-organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGR) that develop severe hypertension, target-organ damage, and die untreated within 7–8 weeks. Recombinant relaxin at a low (26 μg/kg/d) and a high dose (240 μg/kg/d) was given to 4 week-old dTGR and age-matched Sprague-Dawley rats (SD). Systolic blood pressure increased progressively in untreated dTGRs from 162±3 mmHg at week 5 to 225±5 mmHg at week 7. Relaxin had no effect on blood pressure whereas SD rats were normotensive (106±1 mmHg). Untreated and relaxin-treated dTGR had similarly severe cardiac hypertrophy indices. Relaxin did not ameliorate albuminuria and did not prevent matrix-protein deposition in the heart and kidney in dTGR. Finally, relaxin treatment did not reduce mortality. These data suggest that pharmacological doses of relaxin do not reverse severe effects of Ang II. PMID:24710077

  4. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

    PubMed

    Gajula, Kiran S; Huwe, Peter J; Mo, Charlie Y; Crawford, Daniel J; Stivers, James T; Radhakrishnan, Ravi; Kohli, Rahul M

    2014-09-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  5. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  6. Adamantyl-tethered-biphenylic compounds induce apoptosis in cancer cells by targeting Bcl homologs.

    PubMed

    Anusha, Sebastian; Mohan, Chakrabhavi Dhananjaya; Ananda, Hanumappa; Baburajeev, C P; Rangappa, Shobith; Mathai, Jessin; Fuchs, Julian E; Li, Feng; Shanmugam, Muthu K; Bender, Andreas; Sethi, Gautam; Basappa; Rangappa, Kanchugarakoppal S

    2016-02-01

    Bcl homologs prominently contribute to apoptotic resistance in cancer cells and serve as molecular targets in treatment of various cancers. Herein, we report the synthesis of biphenyl-adamantane derivatives by a ligand free palladium on carbon based Suzuki reaction using diisopropylamine as a base for the coupling of adamantane based aryl chloride with a variety of aryl boronic acids. Among the biphenyl derivatives synthesized, compound 3'-(adamantan-1-yl)-4'-methoxy-[1,1'-biphenyl]-3-ol (AMB) displayed cytotoxic activity against hepatocellular carcinoma cell lines without significantly affecting the normal cell lines. Further, AMB caused increased accumulation of the HCC cells in subG1 phase, decreased the expression of Bcl-2, Bcl-xL, cyclin D1, caspase-3, survivin and increased the cleavage of PARP in a time-dependent manner. In silico molecular interaction studies between Bcl homologs and AMB showed that the biphenyl scaffold is predicted to form π-π interactions with Phe-101 and Tyr-105 and the adamantyl fragment is predicted to occupy another hydrophobic region in the kink region of the binding groove. In summary, we report on the synthesis and biological characterization of adamantyl-tethered biphenylic compounds that induce apoptosis in tumor cells most likely by targeting Bcl homologs. PMID:26725030

  7. MIR181A regulates starvation- and rapamycin-induced autophagy through targeting of ATG5

    PubMed Central

    Tekirdag, Kumsal Ayse; Korkmaz, Gozde; Ozturk, Deniz Gulfem; Agami, Reuven; Gozuacik, Devrim

    2013-01-01

    Macroautophagy (autophagy herein) is a cellular catabolic mechanism activated in response to stress conditions including starvation, hypoxia and misfolded protein accumulation. Abnormalities in autophagy were associated with pathologies including cancer and neurodegenerative diseases. Hence, elucidation of the signaling pathways controlling autophagy is of utmost importance. Recently we and others described microRNAs (miRNAs) as novel and potent modulators of the autophagic activity. Here, we describe MIR181A (hsa-miR-181a-1) as a new autophagy-regulating miRNA. We showed that overexpression of MIR181A resulted in the attenuation of starvation- and rapamycin-induced autophagy in MCF-7, Huh-7 and K562 cells. Moreover, antagomir-mediated inactivation of endogenous miRNA activity stimulated autophagy. We identified ATG5 as an MIR181A target. Indeed, ATG5 cellular levels were decreased in cells upon MIR181A overexpression and increased following the introduction of antagomirs. More importantly, overexpression of ATG5 from a miRNA-insensitive cDNA construct rescued autophagic activity in the presence of MIR181A. We also showed that the ATG5 3′ UTR contained functional MIR181A responsive sequences sensitive to point mutations. Therefore, MIR181A is a novel and important regulator of autophagy and ATG5 is a rate-limiting miRNA target in this effect. PMID:23322078

  8. Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus*

    PubMed Central

    Heupel, Wolfgang-Moritz; Müller, Thomas; Efthymiadis, Athina; Schmidt, Enno; Drenckhahn, Detlev; Waschke, Jens

    2009-01-01

    Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment. PMID:19164289

  9. Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging.

    PubMed

    Wang, Xiaoyan; Song, Panshu; Peng, Lu; Tong, Aijun; Xiang, Yu

    2016-01-13

    Conventional fluorophores usually undergo aggregation-caused quenching (ACQ), which limits the loading amount of these fluorophores in nanoparticles for bright fluorescence imaging. On the contrary, fluorophores with aggregation-induced emission (AIE) characteristics are strongly fluorescent in their aggregate states and have been an ideal platform for developing highly fluorescent nanomaterials, such as fluorescent silica nanoparticles (FSNPs). In this work, AIE luminogens based on salicylaldehyde hydrazones were embedded in silica nanoparticles through a facile noncovalent approach, which afforded AIE-FSNPs emitting much brighter fluorescence than that of some commercial fluorescein-doped silica and polystyrene nanoparticles. These AIE-FSNPs displaying multiple fluorescence colors were fabricated by a general method, and they underwent much less fluorescence variation due to environmental pH changes compared with fluorescein-hybridized FSNPs. In addition, a DNA aptamer specific to nucleolin was functionalized on the surface of AIE-FSNPs for targeted cell imaging. Fluorescent microscopy and flow cytometry studies both revealed highly selective fluorescence staining of MCF-7 (a cancer cell line with nucleolin overexpression) over MCF-10A (normal) cells by the aptamer-functionalized AIE-FSNPs. The fluorescence imaging in different color channels was achieved using AIE-FSNPs containing each of the AIE luminogens, as well as photoactivatable fluorescent imaging of target cells by the caged AIE fluorophore. PMID:26653325

  10. Human Induced Pluripotent Stem Cells for Tumor Targeted Delivery of Gold Nanorods and Enhanced Photothermal Therapy.

    PubMed

    Liu, Yanlei; Yang, Meng; Zhang, Jingpu; Zhi, Xiao; Li, Chao; Zhang, Chunlei; Pan, Fei; Wang, Kan; Yang, Yuming; Martinez de la Fuentea, Jesus; Cui, Daxiang

    2016-02-23

    How to improve effective accumulation and intratumoral distribution of plasmonic gold nanoparticles has become a great challenge for photothermal therapy of tumors. Herein, we reported a nanoplatform with photothermal therapeutic effects by fabricating Au nanorods@SiO2@CXCR4 nanoparticles and loading the prepared nanoparticles into the human induced pluripotent stem cells(AuNRs-iPS). In virtue of the prominent optical properties of Au nanorods@SiO2@CXCR4 and remarkable tumor target migration ability of iPS cells, the Au nanorods delivery mediated by iPS cells via the nanoplatform AuNRs-iPS was found to have a prolonged retention time and spatially even distribution in MGC803 tumor-bearing nude mice observed by photoacoustic tomography and two-photon luminescence. On the basis of these improvements, the nanoplatform displayed a robust migration capacity to target the tumor site and to improve photothermal therapeutic efficacy on inhibiting the growth of tumors in xenograft mice under a low laser power density. The combination of gold nanorods with human iPS cells as a theranostic platform paves an alternative road for cancer theranostics and holds great promise for clinical translation in the near future. PMID:26761620

  11. Ultrasound-targeted antisense oligonucleotide attenuates ischemia/reperfusion-induced myocardial tumor necrosis factor-alpha.

    PubMed

    Erikson, John M; Freeman, Gregory L; Chandrasekar, Bysani

    2003-01-01

    Ultrasound contrast agents are now emerging as effective vehicles for delivering therapeutic agents to target tissues. In the present study, we used ultrasound-targeted, contrast-bound antisense oligonucleotides to inhibit the expression of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine with negative inotropic effects. We compared the efficacy of left ventricular vs. intravenous administration and determined the optimal time for delivery. WKY rats were treated with perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microspheres incubated with 100 microg of antisense oligonucleotide directed against TNF-alpha. Contrast was infused into either the superior vena cava or the left ventricular cavity along with simultaneous application of ultrasound. Twenty-four hours later, the animals underwent 15 min of ischemia and 2 h reperfusion. Control animals underwent sham operation only, ischemia/reperfusion only, or received PESDA only. A second group received treatment just prior to, or immediately after the onset of ischemia. At the end of the experimental period, hearts were removed and analyzed for TNF-alpha by northern and western blotting. While no TNF-alpha expression was detected in sham-operated animals, robust expression of TNF-alpha mRNA and protein was seen in controls treated with ultrasound and PESDA alone. In contrast, intravenous or left ventricular administration of antisense oligonucleotides significantly inhibited ischemia/reperfusion-induced TNF-alpha expression. Direct delivery into the left ventricular cavity was more effective than intravenous administration, and delivery just prior to ischemia was most effective in attenuating TNF-alpha expression. Furthermore, attenuation of TNF-alpha expression also significantly inhibited other post-ischemic inflammatory mediators including IL-1beta and intercellular adhesion molecule-1 (ICAM-1). Thus, ultrasound-targeted antisense oligonucleotides can effectively attenuate post

  12. Kdm6b and Pmepa1 as Targets of Bioelectrically and Behaviorally Induced Activin A Signaling.

    PubMed

    Link, Andrea S; Kurinna, Svitlana; Havlicek, Steven; Lehnert, Sandra; Reichel, Martin; Kornhuber, Johannes; Winner, Beate; Huth, Tobias; Zheng, Fang; Werner, Sabine; Alzheimer, Christian

    2016-08-01

    The transforming growth factor-β (TGF-β) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain, the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures, a rodent model of electroconvulsive therapy in humans, were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3, the intracellular effectors of activin signaling, was significantly enriched at the Pmepa1 gene, which encodes a negative feedback regulator of TGF-β signaling in cancer cells, and at the Kdm6b gene, which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings, activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly, physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together, our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression, activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity. PMID:26215835

  13. DC targeting DNA vaccines induce protective and therapeutic antitumor immunity in mice

    PubMed Central

    Wang, Qun; Cao, Wei; Yang, Zhi-Gang; Zhao, Guang-Fa

    2015-01-01

    Background: Anti-CD11c antibodies target to the CD11c receptor that mediates antigen presentation to T cells by dendritic cells (DCs). To exploit these properties for immunization purposes, we obtained DC-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragment (scFv) from N418 (scFvN418), a monoclonal antibody binding the mouse DC-restricted surface molecule CD11c, and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. Methods: Induction of humoral and cellular immune responses and antitumoral activity of the DNA vaccines were tested in transplantable HER2/neu-expressing murine tumor models and in transgenic BALB-neuT mice developing spontaneous Neu-driven mammary carcinomas. Results: Upon injection of the breast tumor cell line D2F2/E2 (stably expressing human wild-type HER2), scFvN418-HER2 immunized mice were protected against tumor growth. Even more important for clinical applications, we were able to substantially slow the growth of implanted D2F2/E2 cells by injection of scFvN418-HER2 conjugates into tumor bearing hosts. The existing tumors were eradicated by treatment with scFvN418-HER2 combined with low-dose cyclophosphamide (CTX), which can make a temporary regulatory T cells (Treg) depletion. What’s more, in combination with the low-dose CTX, vaccination with scFvN418-neu significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice. Conclusion: Our results show that DNA vaccine which targeting of dendritic cells in situ by the means of antibody-antigen conjugates may be a novel way to induce long-lasting antitumor immunity. PMID:26770347

  14. Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Curry, Joseph M; Ko, Ying-Hui; Lin, Zhao; Tuluc, Madalina; Cognetti, David; Birbe, Ruth C; Pribitkin, Edmund; Bombonati, Alessandro; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

    2013-01-01

    Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4

  15. Comparative Analysis of Induced vs. Spontaneous Models of Autoimmune Uveitis Targeting the Interphotoreceptor Retinoid Binding Protein

    PubMed Central

    Chen, Jun; Qian, Haohua; Horai, Reiko; Chan, Chi-Chao; Falick, Yishay; Caspi, Rachel R.

    2013-01-01

    Animal models of autoimmunity to the retina mimic specific features of human uveitis, but no model by itself reproduces the full spectrum of human disease. We compared three mouse models of uveitis that target the interphotoreceptor retinoid binding protein (IRBP): (i) the “classical” model of experimental autoimmune uveitis (EAU) induced by immunization with IRBP; (ii) spontaneous uveitis in IRBP T cell receptor transgenic mice (R161H) and (iii) spontaneous uveitis in Autoimmune Regulator (AIRE)−/− mice. Disease course and severity, pathology and changes in visual function were studied using fundus imaging and histological examinations, optical coherence tomography and electroretinography. All models were on the B10.RIII background. Unlike previously reported, IRBP-induced EAU in B10.RIII mice exhibited two distinct patterns of disease depending on clinical scores developed after onset: severe monophasic with extensive destruction of the retina and rapid loss of visual signal, or lower grade with a prolonged chronic phase culminating after several months in retinal degeneration and loss of vision. R161H and AIRE−/− mice spontaneously developed chronic progressive inflammation; visual function declined gradually as retinal degeneration developed. Spontaneous uveitis in R161H mice was characterized by persistent cellular infiltrates and lymphoid aggregation, whereas AIRE−/− mice characteristically developed multi-focal infiltrates and severe choroidal inflammation. These data demonstrate variability and unique distinguishing features in the different models of uveitis, suggesting that each one can represent distinct aspects of uveitis in humans. PMID:24015215

  16. Functional Food Targeting the Regulation of Obesity-Induced Inflammatory Responses and Pathologies

    PubMed Central

    Hirai, Shizuka; Takahashi, Nobuyuki; Goto, Tsuyoshi; Lin, Shan; Uemura, Taku; Yu, Rina; Kawada, Teruo

    2010-01-01

    Obesity is associated with a low-grade systemic chronic inflammatory state, characterized by the abnormal production of pro- and anti-inflammatory adipocytokines. It has been found that immune cells such as macrophages can infiltrate adipose tissue and are responsible for the majority of inflammatory cytokine production. Obesity-induced inflammation is considered a potential mechanism linking obesity to its related pathologies, such as insulin resistance, cardiovascular diseases, type-2 diabetes, and some immune disorders. Therefore, targeting obesity-related inflammatory components may be a useful strategy to prevent or ameliorate the development of such obesity-related diseases. It has been shown that several food components can modulate inflammatory responses in adipose tissue via various mechanisms, some of which are dependent on peroxisome proliferator-activated receptor γ (PPARγ), whereas others are independent on PPARγ, by attenuating signals of nuclear factor-κB (NF-κB) and/or c-Jun amino-terminal kinase (JNK). In this review, we introduce the beneficial effects of anti-inflammatory phytochemicals that can help prevent obesity-induced inflammatory responses and pathologies. PMID:20508825

  17. Registered report: Intestinal inflammation targets cancer-inducing activity of the microbiota

    PubMed Central

    Eaton, Kate; Yang, Wanwan

    2015-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota’ by Arthur et al. (2012), published in Science in 2012. Arthur and colleagues identified a genotoxic island in Escherichia coli NC101 that appeared to be responsible for causing neoplastic lesions in inflammation-induced IL10−/− mice treated with azoxymethane. The experiments that will be replicated are those reported in Figure 4 (Arthur et al., 2012). Arthur and colleagues inoculated IL10−/− mice with a mutated strain of E. coli NC101 lacking the genotoxic island, and showed that those mice suffered from fewer neoplastic lesions than mice inoculated with the wild type form of E. coli NC101 (Figure 4). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.04186.001 PMID:25781343

  18. Iron overload as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis.

    PubMed

    Toyokuni, Shinya

    2014-01-01

    Few people expected that asbestos, a fibrous mineral, would be carcinogenic to humans. In fact, asbestos is a definite carcinogen in humans, causing a rare but aggressive cancer called malignant mesothelioma (MM). Mesothelial cells line the three somatic cavities and thus do not face the outer surface, but reduce the friction among numerous moving organs. MM has several characteristics: extremely long incubation period of 30-40 years after asbestos exposure, difficulty in clinical diagnosis at an early stage, and poor prognosis even under the current multimodal therapies. In Japan, 'Kubota shock' attracted considerable social attention in 2005 for asbestos-induced mesothelioma and, thereafter, the government enacted a law to provide the people suffering from MM a financial allowance. Several lines of recent evidence suggest that the major pathology associated with asbestos-induced MM is local iron overload, associated with asbestos exposure. Preclinical studies to prevent MM after asbestos exposure with iron reduction are in progress. In addition, novel target genes in mesothelial carcinogenesis have been discovered with recently recognized mesothelioma-prone families. Development of an effective preventive strategy is eagerly anticipated because of the long incubation period for MM. PMID:24257681

  19. A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria

    PubMed Central

    Geng, Ji; Li, Jing; Huang, Tao; Zhao, Kaidi; Chen, Qiuyun; Guo, Wenjie; Gao, Jing

    2016-01-01

    Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin-transferrin receptor system, the present study synthesized and examined the potential use of Adpa-Mn as a novel antitumor agent. Adpa-Mn time and dose-dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa-Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases-9, -7 and -3 and poly (ADP-ribose) polymerase. In addition, Adpa-Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy-related protein microtubule-associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine enhanced Adpa-Mn-induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa-Mn-treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa-Mn-induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa-Mn may target the mitochondria. Taken together, these data suggested that Adpa-Mn may be considered for use as a novel anti-glioma therapeutic option. PMID:27432745

  20. Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

    PubMed Central

    Salmela, Anna-Leena; Pouwels, Jeroen; Varis, Asta; Kukkonen, Anu M.; Toivonen, Pauliina; Halonen, Pasi K.; Perälä, Merja; Kallioniemi, Olli; Gorbsky, Gary J.; Kallio, Marko J.

    2009-01-01

    Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 μg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound. PMID:19395653

  1. Confinement effects of shock waves on laser-induced plasma from a graphite target

    SciTech Connect

    Huang, Feiling; Liang, Peipei; Yang, Xu; Cai, Hua; Wu, Jiada; Xu, Ning; Ying, Zhifeng; Sun, Jian

    2015-06-15

    The spatial confinement effects of shock waves on the laser-induced plasma (LIP) from a graphite target in air were studied by probe beam deflection (PBD) measurements and optical emission spectroscopy (OES). A clear relationship between the confinement of the LIP by the shock wave and the effects on the LIP emission was observed, and the underlying mechanisms are discussed. PBD monitoring revealed that the laser-ablation induced shock wave could be well analogized to the shock wave generated by a point explosion and would be reflected by a block. OES measurements indicated that the optical emission of the LIP exhibited significant variations with the block placement. A first enhancement and then a fast decay of CN molecular emission as well as a suppression of carbon atomic emission were observed in the presence of the block. The results revealed that the reflected shock wave spatially confined the expansion of the LIP and compressed the LIP after encountering it, pushing back the species of the LIP and changing the density of the LIP species including luminous carbon atoms and CN molecules. It is suggested that the change of the LIP emission is attributed to the density variation of the LIP species due to the compression of the LIP and the reactions occurring in the plasma.

  2. Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

    PubMed

    Corso, Lucia; Cavallero, Anna; Baroni, Debora; Garbati, Patrizia; Prestipino, Gianfranco; Bisti, Silvia; Nobile, Mario; Picco, Cristiana

    2016-03-01

    P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases. PMID:26739703

  3. Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells.

    PubMed

    Huang, Guichun; Tao, Leilei; Shen, Sunan; Chen, Longbang

    2016-01-01

    Tumor hypoxia is one of the important features of lung adenocarcinoma. Chemokines might mediate the effects caused by tumor hypoxia. As confirmed in tumor tissue and serum of patients, CC chemokine 28 (CCL28) was the only hypoxia induced chemokine in lung adenocarcinoma cells. CCL28 could promote tube formation, migration and proliferation of endothelial cells. In addition, angiogenesis was promoted by CCL28 in the chick chorioallantoic membrane and matrigel implanted in dorsal back of athymic nude mice (CByJ.Cg-Foxn1(nu)/J). Tumors formed by lung adenocarcinoma cells with high expression of CCL28 grew faster and had a higher vascular density, whereas tumor formation rate of lung adenocarcinoma cells with CCL28 expression knockdown was quite low and had a lower vascular density. CCR3, receptor of CCL28, was highly expressed in vascular endothelial cells in lung adenocarcinoma when examining by immunohistochemistry. Further signaling pathways in endothelial cells, modulated by CCL28, were analyzed by Phosphorylation Antibody Array. CCL28/CCR3 signaling pathway could bypass that of VEGF/VEGFR on the levels of PI3K-Akt, p38 MAPK and PLC gamma. The effects could be neutralized by antibody against CCR3. In conclusion, CCL28, as a chemokine induced by tumor hypoxia, could promote angiogenesis in lung adenocarcinoma through targeting CCR3 on microvascular endothelial cells. PMID:27250766

  4. Registered report: Intestinal inflammation targets cancer-inducing activity of the microbiota.

    PubMed

    Eaton, Kate; Yang, Wanwan

    2015-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota’ by Arthur et al. (2012), published in Science in 2012. Arthur and colleagues identified a genotoxic island in Escherichia coli NC101 that appeared to be responsible for causing neoplastic lesions in inflammation-induced IL10-/- mice treated with azoxymethane. The experiments that will be replicated are those reported in Figure 4 (Arthur et al., 2012). Arthur and colleagues inoculated IL10-/- mice with a mutated strain of E. coli NC101 lacking the genotoxic island, and showed that those mice suffered from fewer neoplastic lesions than mice inoculated with the wild type form of E. coli NC101 (Figure 4). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. PMID:25781343

  5. Targeted expression of cystatin restores fertility in cysteine protease induced male sterile tobacco plants.

    PubMed

    Shukla, Pawan; Subhashini, Mranu; Singh, Naveen Kumar; Ahmed, Israr; Trishla, Shalibhadra; Kirti, P B

    2016-05-01

    Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase-barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops. PMID:26993235

  6. Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells

    PubMed Central

    Huang, Guichun; Tao, Leilei; Shen, Sunan; Chen, Longbang

    2016-01-01

    Tumor hypoxia is one of the important features of lung adenocarcinoma. Chemokines might mediate the effects caused by tumor hypoxia. As confirmed in tumor tissue and serum of patients, CC chemokine 28 (CCL28) was the only hypoxia induced chemokine in lung adenocarcinoma cells. CCL28 could promote tube formation, migration and proliferation of endothelial cells. In addition, angiogenesis was promoted by CCL28 in the chick chorioallantoic membrane and matrigel implanted in dorsal back of athymic nude mice (CByJ.Cg-Foxn1nu/J). Tumors formed by lung adenocarcinoma cells with high expression of CCL28 grew faster and had a higher vascular density, whereas tumor formation rate of lung adenocarcinoma cells with CCL28 expression knockdown was quite low and had a lower vascular density. CCR3, receptor of CCL28, was highly expressed in vascular endothelial cells in lung adenocarcinoma when examining by immunohistochemistry. Further signaling pathways in endothelial cells, modulated by CCL28, were analyzed by Phosphorylation Antibody Array. CCL28/CCR3 signaling pathway could bypass that of VEGF/VEGFR on the levels of PI3K-Akt, p38 MAPK and PLC gamma. The effects could be neutralized by antibody against CCR3. In conclusion, CCL28, as a chemokine induced by tumor hypoxia, could promote angiogenesis in lung adenocarcinoma through targeting CCR3 on microvascular endothelial cells. PMID:27250766

  7. Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

    PubMed Central

    Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

    2013-01-01

    The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

  8. 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

  9. Targeting ALCAM in the cryo-treated tumour microenvironment successfully induces systemic anti-tumour immunity.

    PubMed

    Kudo-Saito, Chie; Fuwa, Takafumi; Kawakami, Yutaka

    2016-07-01

    Cryoablative treatment has been widely used for treating cancer. However, the therapeutic efficacies are still controversial. The molecular mechanisms of the cryo-induced immune responses, particularly underlying the ineffectiveness, remain to be fully elucidated. In this study, we identified a new molecular mechanism involved in the cryo failure. We used cryo-ineffective metastatic tumour models that murine melanoma B16-F10 cells were subcutaneously and intravenously implanted into C57BL/6 mice. When the subcutaneous tumours were treated cryoablation on day 7 after tumour implantation, cells expressing activated leucocyte cell adhesion molecule (ALCAM/CD166) were significantly expanded not only locally in the treated tumours but also systemically in spleen and bone marrow of the mice. The cryo-induced ALCAM(+) cells including CD45(-) mesenchymal stem/stromal cells, CD11b(+)Gr1(+) myeloid-derived suppressor cells, and CD4(+)Foxp3(+) regulatory T cells significantly suppressed interferon γ production and cytotoxicity of tumour-specific CD8(+) T cells via ALCAM expressed in these cells. This suggests that systemic expansion of the ALCAM(+) cells negatively switches host-immune directivity to the tumour-supportive mode. Intratumoural injection with anti-ALCAM blocking monoclonal antibody (mAb) following the cryo treatment systemically induced tumour-specific CD8(+) T cells with higher cytotoxic activities, resulting in suppression of tumour growth and metastasis in the cryo-resistant tumour models. These suggest that expansion of ALCAM(+) cells is a determinant of limiting the cryo efficacy. Further combination with an immune checkpoint inhibitor anti-CTLA4 mAb optimized the anti-tumour efficacy of the dual-combination therapy. Targeting ALCAM may be a promising strategy for overcoming the cryo ineffectiveness leading to the better practical use of cryoablation in clinical treatment of cancer. PMID:27208904

  10. REDD1 Is a Major Target of Testosterone Action in Preventing Dexamethasone-Induced Muscle Loss

    PubMed Central

    Wu, Yong; Zhao, Weidong; Zhao, Jingbo; Zhang, Yuanfei; Qin, Weiping; Pan, Jiangping; Bauman, William A.; Blitzer, Robert D.; Cardozo, Christopher

    2010-01-01

    Glucocorticoids are a well-recognized and common cause of muscle atrophy that can be prevented by testosterone. However, the molecular mechanisms underlying such protection have not been described. Thus, the global effects of testosterone on dexamethasone-induced changes in gene expression were evaluated in rat gastrocnemius muscle using DNA microarrays. Gene expression was analyzed after 7-d administration of dexamethasone, dexamethasone plus testosterone, or vehicle. Dexamethasone changed expression of 876 probe sets by at least 2-fold. Among these, 474 probe sets were changed by at least 2-fold in the opposite direction in the dexamethasone plus testosterone group (genes in opposition). Major biological themes represented by genes in opposition included IGF-I signaling, myogenesis and muscle development, and cell cycle progression. Testosterone completely prevented the 22-fold increase in expression of the mammalian target of rapamycin (mTOR) inhibitor regulated in development and DNA damage responses 1 (REDD1), and attenuated dexamethasone induced increased expression of eIF4E binding protein 1, Forkhead box O1, and the p85 regulatory subunit of the IGF-I receptor but prevented decreased expression of IRS-1. Testosterone attenuated increases in REDD1 protein in skeletal muscle and L6 myoblasts and prevented dephosphorylation of p70S6 kinase at the mTOR-dependent site Thr389 in L6 myoblast cells. Effects of testosterone on REDD1 mRNA levels occurred within 1 h, required the androgen receptor, were blocked by bicalutamide, and were due to inhibition of transcriptional activation of REDD1 by dexamethasone. These data suggest that testosterone blocks dexamethasone-induced changes in expression of REDD1 and other genes that collectively would otherwise down-regulate mTOR activity and hence also down-regulate protein synthesis. PMID:20032058

  11. Control and target gene selection for studies on UV-induced genotoxicity in whales

    PubMed Central

    2013-01-01

    on the capacity of wildlife to resolve or limit UV-induced damage. The proposed target genes are HSP70, P53 and KIN, known to be involved in genotoxic stress pathways, and whose expression patterns can be accurately assessed by using two stable control genes, RPL4 and RPS18. PMID:23837727

  12. Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

    SciTech Connect

    Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu; Duraisamy, Prabhu Kumar; Ashok Kumar, Sekar

    2015-08-28

    The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER{sup +} and ER{sup −} breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen.

  13. Targeting with bovine CD154 enhances humoral immune responses induced by a DNA vaccine in sheep.

    PubMed

    Manoj, Sharmila; Griebel, Philip J; Babiuk, Lorne A; van Drunen Littel-van den Hurk, Sylvia

    2003-01-15

    CD40-CD154 interactions play an important role in regulating humoral and cell-mediated immune responses. Recently, these interactions have been exploited for the development of therapeutic and preventive treatments. The objective of this study was to test the ability of bovine CD154 to target a plasmid-encoded Ag to CD40-expressing APCs. To achieve this, a plasmid coding for bovine CD154 fused to a truncated secreted form of bovine herpesvirus 1 glycoprotein D (tgD), pSLIAtgD-CD154, was constructed. The chimeric tgD-CD154 was expressed in vitro in COS-7 cells and reacted with both glycoprotein D- and CD154-specific Abs. Both tgD and tgD-CD154 were capable of binding to epithelial cells, whereas only tgD-CD154 bound to B cells. Furthermore, dual-labeling of ovine PBMCs revealed that tgD-CD154 was bound by primarily B cells. The functional integrity of the tgD-CD154 chimera was confirmed by the induction of both IL-4-dependent B cell proliferation and tgD-specific lymphoproliferative responses in vitro. Finally, sheep immunized with pSLIAtgD-CD154 developed a more rapid primary tgD-specific Ab response and a significantly stronger tgD-specific secondary response when compared with animals immunized with pSLIAtgD and control animals. Similarly, virus-neutralizing Ab titers were significantly higher after secondary immunization with pSLIAtgD-CD154. These results demonstrate that using CD154 to target plasmid-expressed Ag can significantly enhance immune responses induced by a DNA vaccine. PMID:12517965

  14. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

    PubMed Central

    Yusa, Kosuke; Rashid, S. Tamir; Strick-Marchand, Helene; Varela, Ignacio; Liu, Pei-Qi; Paschon, David E.; Miranda, Elena; Ordóñez, Adriana; Hannan, Nick; Rouhani, Foad Jafari; Darche, Sylvie; Alexander, Graeme; Marciniak, Stefan J.; Fusaki, Noemi; Hasegawa, Mamoru; Holmes, Michael C.; Di Santo, James P.; Lomas, David A.; Bradley, Allan; Vallier, Ludovic

    2011-01-01

    Human induced pluripotent stem cells (hIPSCs) represent a unique opportunity for regenerative medicine since they offer the prospect of generating unlimited quantities of cells for autologous transplantation as a novel treatment for a broad range of disorders1,2,3,4. However, the use of hIPSCs in the context of genetically inherited human disease will require correction of disease-causing mutations in a manner that is fully compatible with clinical applications3,5. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome6. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of hIPSCs. Here, we show that a combination of zinc finger nucleases (ZFNs)7 and piggyBac8,9 technology in hIPSCs can achieve bi-allelic correction of a point mutation (Glu342Lys) in the α1-antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α1-antitrypsin deficiency (A1ATD). Genetic correction of hIPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle for the potential of combining hIPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies. PMID:21993621

  15. Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

    PubMed Central

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-01-01

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

  16. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    PubMed

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-01-01

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

  17. Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

    PubMed Central

    Kang, Jeffrey C; Poovassery, Jayakumar S; Bansal, Pankaj; You, Sungyong; Manjarres, Isabel M; Ober, Raimund J; Ward, E Sally

    2014-01-01

    The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert. PMID:24492289

  18. Update of Targeted Therapy-Induced Hypertension: Basics for Non-Oncology Providers.

    PubMed

    Escalante, Carmen P; Lu, Maggie; Marten, Claire A

    2016-01-01

    Over the past several years, cancer treatments have expanded from usual chemotherapy standards with introduction of newer targeted therapies. As with chemotherapy, the targeted therapies also have unique side effects affecting various organ systems producing toxicities, such as cardiac and renal. This manuscript focuses on hypertension induced by vascular endothelial growth factor (VEGF) inhibitors and tyrosine kinase inhibitors (TKI). Hypertension due to these cancer therapies is important because these agents are now frequently used in common cancers. In addition, patients with cancer may not be treated in a comprehensive cancer center with experts available to manage the cancer and other side effects either from the malignancy or treatment of the malignancy. Especially in rural areas, patients are often managed or co-managed by a primary care provider with input from an oncologist that may not be nearby. Our aim is to provide an overview of the latest Federal Drug Administration (FDA) approved VEGF inhibitors and TKI's causing hypertension so that others managing patients on these treatments may easily recognize hypertension attributable to these agents and feel comfortable and confident in providing appropriate management and treatment of this side effect. This update includes characteristics, such as mechanism of action, metabolism and route of administration, and management and treatment of hypertension with aspects such as the timing, duration and monitoring of these agents. In addition, an algorithm for monitoring and treating hypertension before, during and after treatment with these therapies is included. It is imperative for patients to have hypertension promptly treated to prevent complications so they may continue with these agents with the least interruption or discontinuation of treatment, ensuring the best benefit available in their cancer trajectory. PMID:26931476

  19. Exposure-dependent variation in cryolite induced lethality in the non-target insect, Drosophila melanogaster.

    PubMed

    Podder, Sayanti; Roy, Sumedha

    2014-03-01

    The starting point of toxicity testing of any chemical in an organism is the determination of its Lethal Concentration 50 (LC50). In the present study, LC50 of a fluorinated insecticide cryolite is determined in a non-target insect model, Drosophila melanogaster. Interestingly, the result shows that acute LC50 of cryolite was much greater in comparison to the chronic one in case of Drosophila larvae. Larvae which were exposed to 65,000 to 70,000 µg/ml cryolite through food showed 50% mortality after 18 hours of acute exposure, whereas only 150 to 160 µg/ml cryolite was sufficient to cause 50% mortality in case of chronic exposure. Thus cryolite in a small amount when applied once cannot produce noticeable changes in Drosophila, whereas the same amount when used continuously can be fatal. The non-feeding pupal stage was also seen to be affected by chemical treatment. This suggests that the test chemical affects the developmental fate and results in failure of adult emergence. Absence of chemical-induced mortality in adults assumes that the toxicity of cryolite might be restricted to the preimaginal stages of the organism. Reduction in body size of larvae after ingestion of cryolite (with food) in acute treatment schedule is another interesting finding of this study. Some individuals consuming cryolite containing food cannot survive whereas the few survivors manifest a significant growth retardation which might be due to a tendency of refusal in feeding. Hence the present findings provide a scope of assessment of risk of other similar non-target groups. PMID:26038671

  20. Exposure-dependent variation in cryolite induced lethality in the non-target insect, Drosophila melanogaster

    PubMed Central

    Podder, Sayanti

    2014-01-01

    The starting point of toxicity testing of any chemical in an organism is the determination of its Lethal Concentration 50 (LC50). In the present study, LC50 of a fluorinated insecticide cryolite is determined in a non-target insect model, Drosophila melanogaster. Interestingly, the result shows that acute LC50 of cryolite was much greater in comparison to the chronic one in case of Drosophila larvae. Larvae which were exposed to 65,000 to 70,000 µg/ml cryolite through food showed 50% mortality after 18 hours of acute exposure, whereas only 150 to 160 µg/ml cryolite was sufficient to cause 50% mortality in case of chronic exposure. Thus cryolite in a small amount when applied once cannot produce noticeable changes in Drosophila, whereas the same amount when used continuously can be fatal. The non-feeding pupal stage was also seen to be affected by chemical treatment. This suggests that the test chemical affects the developmental fate and results in failure of adult emergence. Absence of chemical-induced mortality in adults assumes that the toxicity of cryolite might be restricted to the preimaginal stages of the organism. Reduction in body size of larvae after ingestion of cryolite (with food) in acute treatment schedule is another interesting finding of this study. Some individuals consuming cryolite containing food cannot survive whereas the few survivors manifest a significant growth retardation which might be due to a tendency of refusal in feeding. Hence the present findings provide a scope of assessment of risk of other similar non-target groups. PMID:26038671

  1. Probing of medium-scale traveling ionospheric disturbances using HF-induced scatter targets

    NASA Astrophysics Data System (ADS)

    Blagoveshchenskaya, N. F.; Borisova, T. D.; Kornienko, V. A.; Moskvin, I. V.; Rietveld, M. T.; Frolov, V. L.; Uryadov, V. P.; Kagan, L. M.; Yampolski, Yu. M.; Galushko, V. L.; Koloskov, A. V.; Kasheev, S. B.; Zalizovski, A. V.; Vertogradov, G. G.; Vertogradov, V. G.; Kelley, M. C.

    2006-09-01

    Experimental results from the Tromsø and Sura heating experiments at high and mid-latitudes are examined. It is shown that the combination of HF-induced target and bi-static HF Doppler radio scatter observations is a profitable method for probing medium-scale traveling ionospheric disturbances (TIDs) at high and mid-latitudes. HF ionospheric modification experiments provide a way of producing the HF-induced scatter target in a controlled manner at altitudes where the sensitivity to TIDs is highest. Bi-static HF Doppler radio scatter observations were carried out on the London-Tromsø-St. Petersburg path in the course of a Tromsø heating experiment on 16 November 2004 when the pump wave was reflected from an auroral Es-layer. During Sura heating experiments on 19 and 20 August 2004, when the HF pump wave was reflected from the F2 ionospheric layer, multi-position bi-static HF Doppler radio scatter observations were simultaneously performed at three reception points including St. Petersburg, Kharkov, and Rostov-on-Don. Ray tracing and Doppler shift simulations were made for all experiments. A computational technique has been developed allowing the reconstruction of the TID phase velocities from multi-position bi-static HF Doppler scatters. Parameters of medium-scale TIDs were found. In all experiments they were observed in the evening and pre-midnight hours. TIDs in the auroral E-region with periods of about 23 min were traveling southward at speeds of 210 m/s. TIDs in the mid-latitudinal F-region with periods from 20 to 45 min travelled at speeds between 40 and 150 m/s. During quiet magnetic conditions the waves were traveling in the north-east direction. In disturbed conditions the waves were moving in the south-west direction with higher speeds as compared with quiet conditions. Possible sources for the atmospheric gravity waves at middle and high latitudes are discussed.

  2. The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish.

    PubMed

    White, David T; Mumm, Jeff S

    2013-08-15

    At the turn of the 20th century, classical regenerative biology--the study of organismal/tissue/limb regeneration in animals such as crayfish, snails, and planaria--garnered much attention. However, scientific luminaries such as Thomas Hunt Morgan eventually turned to other fields after concluding that inquiries into regenerative mechanisms were largely intractable beyond observational intrigues. The field of regeneration has enjoyed a resurgence in research activity at the turn of the 21st century, in large part due to "the promise" of cultured stem cells regarding reparative therapeutic approaches. Additionally, genomics-based methods that allow sophisticated genetic/molecular manipulations to be carried out in nearly any species have extended organismal regenerative biology well beyond observational limits. Throughout its history, complex paradigms such as limb regeneration--involving multiple tissue/cell types, thus, potentially multiple stem cell subtypes--have predominated the regenerative biology field. Conversely, cellular regeneration--the replacement of specific cell types--has been studied from only a few perspectives (predominantly muscle and mechanosensory hair cells). Yet, many of the degenerative diseases that regenerative biology hopes to address involve the loss of individual cell types; thus, a primary emphasis of the embryonic/induced stem cell field is defining culture conditions which promote cell-specific differentiation. Here we will discuss recent methodological approaches that promote the study of cell-specific regeneration. Such paradigms can reveal how the differentiation of specific cell types and regenerative potential of discrete stem cell niches are regulated. In particular, we will focus on how the nitroreductase (NTR) system of inducible targeted cell ablation facilitates: (1) large-scale genetic and chemical screens for identifying factors that regulate regeneration and (2) in vivo time-lapse imaging experiments aimed at

  3. Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

    PubMed Central

    Bonacina, F.; Barbieri, S.S.; Cutuli, L.; Amadio, P.; Doni, A.; Sironi, M.; Tartari, S.; Mantovani, A.; Bottazzi, B.; Garlanda, C.; Tremoli, E.; Catapano, A.L.; Norata, G.D.

    2016-01-01

    Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cell's derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis. PMID:26976330

  4. IL-2/IL-2 Ab Therapy Induces Target Organ NK Cells that Inhibit CNS Inflammation

    PubMed Central

    Hao, Junwei; Campagnolo, Denise; Liu, Ruolan; Piao, Wenhua; Shi, Samuel; Hu, Baoyong; Xiang, Rong; Zhou, Qinghua; Vollmer, Timothy; Van Kaer, Luc; La Cava, Antonio; Shi, Fu-Dong

    2010-01-01

    Objective The role of natural killer (NK) cells in regulating multiple sclerosis (MS) is not well understood. Additional studies with NK cells might provide insight into the mechanism of action of MS therapies such as daclizumab, an antibody against the IL-2R α-chain, which induces expansion of CD56bright NK cells. Methods In a relapsing-remitting form of the experimental autoimmune encephalomyelitis (EAE) model of MS induced in SJL mice, we expanded NK cells with IL-2 coupled with an anti-IL-2 mAb and evaluated the effects of these NK cells on EAE. Further, we investigated the effect of the human version of IL-2/IL-2 mAb on NK cells from MS patients and its effect on CNS inflammation and pathology in a human-mouse chimera model and assessed the underlying mechanisms. Results IL-2/IL-2 mAb dramatically expands NK cells both in the peripheral lymphoid organs and in the central nervous system (CNS), and attenuates CNS inflammation and neurological deficits. Disease protection is conferred by CNS-resident NK cells. Importantly, the human version of IL-2/IL-2 mAb restored the defective CD56+ NK cells from MS patients in a human-mouse chimera model. Both the CD56bright and CD56dim subpopulations were required to attenuate disease in this model. Interpretation These findings unveil the immunotherapeutic potential of NK cells, which can act as critical suppressor cells in target organs of autoimmunity. These results also have implications to better understand the mechanism of action of daclizumab in MS. PMID:21425186

  5. α-Synuclein-induced myelination deficit defines a novel interventional target for multiple system atrophy.

    PubMed

    Ettle, Benjamin; Kerman, Bilal E; Valera, Elvira; Gillmann, Clarissa; Schlachetzki, Johannes C M; Reiprich, Simone; Büttner, Christian; Ekici, Arif B; Reis, André; Wegner, Michael; Bäuerle, Tobias; Riemenschneider, Markus J; Masliah, Eliezer; Gage, Fred H; Winkler, Jürgen

    2016-07-01

    Multiple system atrophy (MSA) is a rare atypical parkinsonian disorder characterized by a rapidly progressing clinical course and at present without any efficient therapy. Neuropathologically, myelin loss and neurodegeneration are associated with α-synuclein accumulation in oligodendrocytes, but underlying pathomechanisms are poorly understood. Here, we analyzed the impact of oligodendrocytic α-synuclein on the formation of myelin sheaths to define a potential interventional target for MSA. Post-mortem analyses of MSA patients and controls were performed to quantify myelin and oligodendrocyte numbers. As pre-clinical models, we used transgenic MSA mice, a myelinating stem cell-derived oligodendrocyte-neuron co-culture, and primary oligodendrocytes to determine functional consequences of oligodendrocytic α-synuclein overexpression on myelination. We detected myelin loss accompanied by preserved or even increased numbers of oligodendrocytes in post-mortem MSA brains or transgenic mouse forebrains, respectively, indicating an oligodendrocytic dysfunction in myelin formation. Corroborating this observation, overexpression of α-synuclein in primary and stem cell-derived oligodendrocytes severely impaired myelin formation, defining a novel α-synuclein-linked pathomechanism in MSA. We used the pro-myelinating activity of the muscarinic acetylcholine receptor antagonist benztropine to analyze the reversibility of the myelination deficit. Transcriptome profiling of primary pre-myelinating oligodendrocytes demonstrated that benztropine readjusts myelination-related processes such as cholesterol and membrane biogenesis, being compromised by oligodendrocytic α-synuclein. Additionally, benztropine restored the α-synuclein-induced myelination deficit of stem cell-derived oligodendrocytes. Strikingly, benztropine also ameliorated the myelin deficit in transgenic MSA mice, resulting in a prevention of neuronal cell loss. In conclusion, this study defines the α-synuclein-induced

  6. Identification of novel therapeutic targets in the secretome of ionizing radiation‑induced senescent tumor cells.

    PubMed

    Hwang, Hyun Jung; Jung, Seung Hee; Lee, Hyung Chul; Han, Na Kyung; Bae, In Hwa; Lee, Minyoung; Han, Young-Hoon; Kang, Young-Sun; Lee, Su-Jae; Park, Heon Joo; Ko, Young-Gyu; Lee, Jae-Seon

    2016-02-01

    Cellular senescence is a state of irreversible growth arrest that can be triggered by multiple mechanisms, including telomere shortening, the epigenetic derepression of the INK4α/ARF locus and DNA damage. Senescence has been considered a tumor‑suppressing mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells have deleterious effects on the tissue microenvironment. Some of these effects could be attributed to the senescence‑associated secretory phenotype that has the ability to promote tumor progression. However, secreted proteins from senescent tumor cells and their effects on the tumor microenvironment due to ionizing radiation (IR) exposure have not yet been fully elucidated. In the present study, we analyzed cytokines secreted from IR‑induced senescent MCF7 cells by using cytokine microarrays and confirmed by western blot analysis that increased secretion of osteoprotegerin (OPG), midkine (MDK) and apolipoprotein E3 (ApoE3) occurs in these cells. Invasive, migratory and wound‑healing activities were observed in MDA‑MB‑231 and MCF‑10A cells following treatment with recombinant human OPG, MDK and ApoE3 proteins. Additionally, tube‑formation activity was assessed in OPG‑, MDK‑ and ApoE3‑treated human umbilical vein endothelial cells (HUVECs). We found that OPG, MDK and ApoE3 affected cell motility and tube‑formation activity. Since OPG markedly affected cell motility, we examined the effect of senescent conditioned media containing neutralizing OPG antibodies on migration and wound‑healing activity. Our results demonstrated that IR‑induced senescent tumor cells influence the tumor microenvironment by increasing the production of cytokines, such as OPG, MDK and ApoE3. Furthermore, these data suggest that OPG is likely a promising target capable of reducing the deleterious effects on the tumor microenvironment during radiation therapy. PMID:26717900

  7. Amelioration of Cisplatin-Induced Experimental Peripheral Neuropathy by a Small Molecule Targeting p75NTR

    PubMed Central

    Friesland, Amy; Weng, Zhiying; Duenas, Maria; Massa, Stephen M.; Longo, Frank M.; Lu, Qun

    2014-01-01

    Cisplatin is an effective and widely used first-line chemotherapeutic drug for treating cancers. However, many patients sustain cisplatin-induced peripheral neuropathy (CIPN), often leading to a reduction in drug dosages or complete cessation of treatment altogether. Therefore, it is important to understand cisplatin mechanisms in peripheral nerve tissue mediating its toxicity and identify signaling pathways for potential intervention. Rho GTPase activation is increased following trauma in several models of neuronal injury. Thus, we investigated whether components of the Rho signaling pathway represent important neuroprotective targets with the potential to ameliorate CIPN and thereby optimize current chemotherapy treatment regimens. We have developed a novel CIPN model in the mouse. Using this model and primary neuronal culture, we determined whether LM11A-31, a small-molecule, orally bioavailable ligand of the p75 neurotrophin receptor (p75NTR), can modulate Rho GTPase signaling and reduce CIPN. Von Frey filament analysis of sural nerve function showed that LM11A-31 treatment prevented decreases in peripheral nerve sensation seen with cisplatin treatment. Morphometric analysis of harvested sural nerves revealed that cisplatin-induced abnormal nerve fiber morphology and the decreases in fiber area were alleviated with concurrent LM11A-31 treatment. Cisplatin treatment increased RhoA activity accompanied by the reduced tyrosine phosphorylation of SHP-2, which was reversed by LM11A-31. LM11A-31 also countered the effects of calpeptin, which activated RhoA by inhibiting SHP-2 tyrosine phosphatase. Therefore, suppression of RhoA signaling by LM11A-31 that blocks proNGF binding to p75NTR or activates SHP-2 tyrosine phosphatase downstream of NGF receptor enhances neuroprotection in experimental CIPN in mouse model. PMID:25277379

  8. A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria.

    PubMed

    Geng, Ji; Li, Jing; Huang, Tao; Zhao, Kaidi; Chen, Qiuyun; Guo, Wenjie; Gao, Jing

    2016-09-01

    Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin‑transferrin receptor system, the present study synthesized and examined the potential use of Adpa‑Mn as a novel antitumor agent. Adpa‑Mn time and dose‑dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa‑Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases‑9, ‑7 and ‑3 and poly (ADP‑ribose) polymerase. In addition, Adpa‑Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy‑related protein microtubule‑associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3‑methyladenine and chloroquine enhanced Adpa‑Mn‑induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa‑Mn‑treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa‑Mn‑induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa‑Mn may target the mitochondria. Taken together, these data suggested that Adpa‑Mn may be considered for use as a novel anti‑glioma therapeutic option. PMID:27432745

  9. Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence*

    PubMed Central

    Hisatomi, Osamu; Nakatani, Yoichi; Takeuchi, Ken; Takahashi, Fumio; Kataoka, Hironao

    2014-01-01

    Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage sensing (LOV) domain at the C terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (full-length, FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide linkages at Cys162 and Cys182 (Hisatomi, O., Takeuchi, K., Zikihara, K., Ookubo, Y., Nakatani, Y., Takahashi, F., Tokutomi, S., and Kataoka, H. (2013) Plant Cell Physiol. 54, 93–106). In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine to serine-mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately −245 ± 15 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2·DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor. PMID:24790107

  10. Whole Brain Radiation-Induced Cognitive Impairment: Pathophysiological Mechanisms and Therapeutic Targets

    PubMed Central

    Lee, Yong Woo; Cho, Hyung Joon; Lee, Won Hee; Sonntag, William E.

    2012-01-01

    Radiation therapy, the most commonly used for the treatment of brain tumors, has been shown to be of major significance in tu-mor control and survival rate of brain tumor patients. About 200,000 patients with brain tumor are treated with either partial large field or whole brain radiation every year in the United States. The use of radiation therapy for treatment of brain tumors, however, may lead to devastating functional deficits in brain several months to years after treatment. In particular, whole brain radiation therapy results in a significant reduction in learning and memory in brain tumor patients as long-term consequences of treatment. Although a number of in vitro and in vivo studies have demonstrated the pathogenesis of radiation-mediated brain injury, the cel-lular and molecular mechanisms by which radiation induces damage to normal tissue in brain remain largely unknown. Therefore, this review focuses on the pathophysiological mechanisms of whole brain radiation-induced cognitive impairment and the iden-tification of novel therapeutic targets. Specifically, we review the current knowledge about the effects of whole brain radiation on pro-oxidative and pro-inflammatory pathways, matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) system and extracellular matrix (ECM), and physiological angiogenesis in brain. These studies may provide a foundation for defin-ing a new cellular and molecular basis related to the etiology of cognitive impairment that occurs among patients in response to whole brain radiation therapy. It may also lead to new opportunities for therapeutic interventions for brain tumor patients who are undergoing whole brain radiation therapy. PMID:24009822

  11. MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

    PubMed Central

    Bild, Matthias; Jung, Ulrike; Müller, Henrik; Arntzen, Magnus Ø.; Piso, Chloe; Stephan, Carsten; Thiede, Bernd; Mollenkopf, Hans-Joachim; Jung, Klaus; Kaufmann, Stefan H. E.; Schreiber, Jörg

    2012-01-01

    Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis. PMID:22532850

  12. Gold nanoparticle plasmonics enhanced ultrafast laser-induced optoporation and stimulation of targeted cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Meunier, Michel; Bergeron, Éric; Lavoie-Cardinal, Flavie; Boutopoulos, Christos; Salesse, Charleen; Winnik, Françoise M.; De Koninck, Paul

    2016-03-01

    Gold nanoparticles (AuNPs) have found numerous applications in nanomedicine in view of their robustness, ease of functionalization and low toxicity. Upon irradiation of AuNPs by a pulsed ultrafast laser, various highly localized phenomena can be obtained including a temperature rise, pressure wave, charge injection and production of nanobubbles close to the cellular membrane [1]. These phenomena can be used to manipulate, optoperforate, transfect and stimulate targeted cells [2-5]. Irradiating at 800 nm in the optically biological transparent window, we demonstrated local optoporation and transfection of cells as well as local stimulation of neurons. Two recent examples will be given: (i) Laser-induced selective optoporation of cells: The technique can be used on various types of cells and a proof of principle will be given on human cancer cells in a co-culture using functionalized AuNPs [6]. (ii) Laser-induced stimulation of neurons and monitoring of the localized Ca2+ signaling: This all optical method uses a standard confocal microscope to trigger a transient increase in free Ca2+ in neurons covered by functionalized AuNPs as well as to measure these local variations optically with the Ca2+ sensor GCaMP6s [7]. The proposed techniques provide a new complement to light-dependent methods in neuroscience. REFERENCES (by our group): (1) Boulais, J. Photochem. Photobiol. C Photochem. Rev. 17, 26 (2013); (2) Baumgart, Biomaterials 33, 2345 (2012); (3) Boulais, NanoLett. 12, 4763 (2012); (4) Boutopoulos, J. Biophotonics (2015); (5) Boutopoulos, Nanoscale 7, 11758 (2015); (6) Bergeron, Biomaterials, submitted (2015); (7) Lavoie-Cardinal, Nature Commun. submitted (2015).

  13. Therapeutic expression of hairpins targeting apolipoprotein B100 induces phenotypic and transcriptome changes in murine liver

    PubMed Central

    Maczuga, P; Verheij, J; van der Loos, C; van Logtenstein, R; Hooijer, G; Martier, R; Borel, F; Lubelski, J; Koornneef, A; Blits, B; van Deventer, S; Petry, H; Konstantinova, P

    2014-01-01

    Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy. PMID:24152580

  14. Characterization of ERK Docking Domain Inhibitors that Induce Apoptosis by Targeting Rsk-1 and Caspase-9

    PubMed Central

    2011-01-01

    proteins. Conclusion These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins. PMID:21219631

  15. Sulphoraphane, a naturally occurring isothiocyanate induces apoptosis in breast cancer cells by targeting heat shock proteins

    SciTech Connect

    Sarkar, Ruma; Mukherjee, Sutapa; Biswas, Jaydip; Roy, Madhumita

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer HSPs (27, 70 and 90) and HSF1 are overexpressed in MCF-7 and MDA-MB-231 cells. Black-Right-Pointing-Pointer Sulphoraphane, a natural isothiocyanate inhibited HSPs and HSF1 expressions. Black-Right-Pointing-Pointer Inhibition of HSPs and HSF1 lead to regulation of apoptotic proteins. Black-Right-Pointing-Pointer Alteration of apoptotic proteins activate of caspases particularly caspase 3 and 9 leading to induction of apoptosis. Black-Right-Pointing-Pointer Alteration of apoptotic proteins induce caspases leading to induction of apoptosis. -- Abstract: Heat shock proteins (HSPs) are involved in protein folding, aggregation, transport and/or stabilization by acting as a molecular chaperone, leading to inhibition of apoptosis by both caspase dependent and/or independent pathways. HSPs are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion and metastasis. HSPs particularly 27, 70, 90 and the transcription factor heat shock factor1 (HSF1) play key roles in the etiology of breast cancer and can be considered as potential therapeutic target. The present study was designed to investigate the role of sulphoraphane, a natural isothiocyanate on HSPs (27, 70, 90) and HSF1 in two different breast cancer cell lines MCF-7 and MDA-MB-231 cells expressing wild type and mutated p53 respectively, vis-a-vis in normal breast epithelial cell line MCF-12F. It was furthermore investigated whether modulation of HSPs and HSF1 could induce apoptosis in these cells by altering the expressions of p53, p21 and some apoptotic proteins like Bcl-2, Bax, Bid, Bad, Apaf-1 and AIF. Sulphoraphane was found to down-regulate the expressions of HSP70, 90 and HSF1, though the effect on HSP27 was not pronounced. Consequences of HSP inhibition was upregulation of p21 irrespective of p53 status. Bax, Bad, Apaf-1, AIF were upregulated followed by down-regulation of Bcl-2 and this effect was prominent

  16. CD19 and CD20 Targeted Vectors Induce Minimal Activation of Resting B Lymphocytes

    PubMed Central

    Schwenkert, Michael; Cosset, François-Loic; Verhoeyen, Els; Buchholz, Christian J.

    2013-01-01

    B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral

  17. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1.

    PubMed

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, KeWei; Lai, Ren

    2015-01-01

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery. PMID:26420335

  18. Influence of electronic stopping on sputtering induced by cluster impact on metallic targets

    SciTech Connect

    Sandoval, Luis; Urbassek, Herbert M.

    2009-04-01

    Using molecular-dynamics simulation, we model the sputtering of a Au (111) crystallite induced by the impact of Au{sub 13} projectiles with total energies up to 500 keV. Due to the uncertainty of the electronic stopping of Au moving in particular at small velocities, we performed several simulations, in which the electronic stopping parameters are systematically changed. Our results demonstrate the dominating influence of the cut-off energy E{sub c}, below which the high-velocity electronic stopping of atoms is switched off in the simulation. If E{sub c} is smaller than roughly one half the cohesive energy of the target, sputtering ceases after a few ps; the spike contribution to sputtering (also called phase explosion or gas-flow contribution) is entirely quenched and the sputtering yield is up to an order of magnitude smaller than when electronic stopping is taken into account only at higher atom energies. Our results demonstrate the importance of a careful modeling of electronic stopping in simulations of spike sputtering from metals.

  19. Hypoxia-Inducible Factor-1 (HIF-1): A Potential Target for Intervention in Ocular Neovascular Diseases

    PubMed Central

    Vadlapatla, Ramya Krishna; Vadlapudi, Aswani Dutt; Mitra, Ashim K.

    2015-01-01

    Constant oxygen supply is essential for proper tissue development, homeostasis and function of all eukaryotic organisms. Cellular response to reduced oxygen levels is mediated by the transcriptional regulator hypoxia-inducible factor-1 (HIF-1). It is a heterodimeric complex protein consisting of an oxygen dependent subunit (HIF-1α) and a constitutively expressed nuclear subunit (HIF-1β). In normoxic conditions, de novo synthesized cytoplasmic HIF-1α is degraded by 26S proteasome. Under hypoxic conditions, HIF-1α is stabilized, binds with HIF-1β and activates transcription of various target genes. These genes play a key role in regulating angiogenesis, cell survival, proliferation, chemotherapy, radiation resistance, invasion, metastasis, genetic instability, immortalization, immune evasion, metabolism and stem cell maintenance. This review highlights the importance of hypoxia signaling in development and progression of various vision threatening pathologies such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration and glaucoma. Further, various inhibitors of HIF-1 pathway that may have a viable potential in the treatment of oxygen-dependent ocular diseases are also discussed. PMID:23701276

  20. Mitochondrially targeted wild-type p53 induces apoptosis in a solid human tumor xenograft model

    PubMed Central

    Palacios, Gustavo; Crawford, Howard C.; Vaseva, Angelina; Moll, Ute M.

    2013-01-01

    Classic but also novel roles of p53 are becoming increasingly well characterized. We previously showed that ex vivo retroviral transfer of mitochondrially targeted wild type p53 (mitop53) in the Eμ-myc mouse lymphoma model efficiently induces tumor cell killing in vivo. In an effort to further explore the therapeutic potential of mitop53 for its pro-apoptotic effect in solid tumors, we generated replication-deficient recombinant human Adenovirus type 5 vectors. We show here that adenoviral delivery of mitop53 by intratumoral injection into HCT116 human colon carcinoma xenograft tumors in nude mice is surprisingly effective, resulting in tumor cell death of comparable potency to conventional p53. These apoptotic effects in vivo were confirmed by Ad5-mitop53 mediated cell death of HCT116 cells in culture. Together, these data provide encouragement to further explore the potential for novel mitop53 proteins in cancer therapy to execute the shortest known circuitry of p53 death signaling. PMID:18719383

  1. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1

    PubMed Central

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, KeWei; Lai, Ren

    2015-01-01

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx–TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery. PMID:26420335

  2. Stressed to Death: Targeting Endoplasmic Reticulum Stress Response Induced Apoptosis in Gliomas

    PubMed Central

    Johnson, Guyla G.; White, Misti C.; Grimaldi, Maurizio

    2012-01-01

    Glial tumors are the main primary adult brain tumor. Even with the most advanced treatments, which include stereotactic microscope aided surgical resection, internal and external radiation therapy and local and systemic chemotherapy, median survival time for patients diagnosed with these malignancies is about 12 months. We explore here the possibility that the endoplasmic reticulum stress response (ERSR) could be a possible target to develop chemotherapeutic agents to induce toxicity in glioma cells. ERSR has the dual capacity of activating repair and/or cytotoxic mechanisms. ERSR is triggered by the accumulation of unfolded proteins in the ER. The presence of unfolded proteins in the ER regulates, via a complex biochemical cascade, the upregulation of molecular chaperones, inhibition of protein synthesis, and an increase of proteasome mediated unfolded protein degradation. ERSR in particular conditions can also contribute to cell death via activation of programmed cell death. Apoptosis activation during ERSR is usually caused by the activation of one or a combination of three biochemical cascades. Induction of these pathways ultimately leads to caspase 3 activation culminating in apoptosis. Glioma cells are in a condition of constant low grade ERSR, which possibly contributes to their resistance to treatment protocols. It is conceivable that small molecules that interact with this phenomenon ultimately could be used to modulate the system to activate apoptosis and cause gliotoxicity. We will discuss here ERSR biochemically relevant features to death mechanisms and already identified small molecules that by modulating ERSR are able to activate glioma cell death. PMID:21348829

  3. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1

    NASA Astrophysics Data System (ADS)

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, Kewei; Lai, Ren

    2015-09-01

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.

  4. Sleep and Pregnancy-Induced Hypertension: A Possible Target for Intervention?

    PubMed Central

    Haney, Alyssa; Buysse, Daniel J.; Okun, Michele

    2013-01-01

    Sleep disturbances in the general population are associated with elevated blood pressure. This may be due to several mechanisms, including sympathetic activation and hypothalamic-pituitary-adrenal (HPA) axis disturbance. Elevated blood pressure in pregnancy can have devastating effects on both maternal and fetal health and is associated with increased risk for preeclampsia and poor delivery outcomes. Preliminary evidence suggests that mechanisms linking sleep and blood pressure in the general population may also hold in the pregnant population. However, the effects of disturbed sleep on physiologic mechanisms that may directly influence blood pressure in pregnancy have not been well studied. The role that sleep disturbance plays in gestational blood pressure elevation and its subsequent consequences warrant further investigation. This review evaluates the current literature on sleep disturbance and elevated blood pressure in pregnancy and proposes possible treatment interventions. Citation: Haney A; Buysse DJ; Okun M. Sleep and pregnancy-induced hypertension: a possible target for intervention? J Clin Sleep Med 2013;9(12):1349-1356. PMID:24340300

  5. TARGETED DELETION OF INDUCIBLE HEAT SHOCK PROTEIN 70 ABROGATES THE LATE INFARCT-SPARING EFFECT OF MYOCARDIAL ISCHEMIC PRECONDITIONING

    EPA Science Inventory

    Abstract submitted for 82nd annual meeting of the American Association for Thoracic Surgery, May 4-8, 2002 in Washington D.C.

    Targeted Deletion of Inducible Heat Shock Protein 70 Abrogates the Late Infarct-Sparing Effect of Myocardial Ischemic Preconditioning

    Craig...

  6. Selective targeting of BCL6 induces oncogene addiction switching to BCL2 in B-cell lymphoma

    PubMed Central

    Patel, Jayeshkumar; Hatzi, Katerina; Malik, Alka; Tam, Wayne; Martin, Peter; Leonard, John; Melnick, Ari; Cerchietti, Leandro

    2016-01-01

    The BCL6 oncogene plays a crucial role in sustaining diffuse large B-cell lymphomas (DLBCL) through transcriptional repression of key checkpoint genes. BCL6-targeted therapy kills lymphoma cells by releasing these checkpoints. However BCL6 also directly represses several DLBCL oncogenes such as BCL2 and BCL-XL that promote lymphoma survival. Herein we show that DLBCL cells that survive BCL6-targeted therapy induce a phenomenon of “oncogene-addiction switching” by reactivating BCL2-family dependent anti-apoptotic pathways. Thus, most DLBCL cells require concomitant inhibition of BCL6 and BCL2-family members for effective lymphoma killing. Moreover, in DLBCL cells initially resistant to BH3 mimetic drugs, BCL6 inhibition induces a newly developed reliance on anti-apoptotic BCL2-family members for survival that translates in acquired susceptibility to BH3 mimetic drugs ABT-737 and obatoclax. In germinal center B cell-like (GCB)-DLBCL cells, the proteasome inhibitor bortezomib and the NEDD inhibitor MLN4924 post-transcriptionally activated the BH3-only sensitizer NOXA thus counteracting the oncogenic switch to BCL2 induced by BCL6-targeting. Hence our study indicates that BCL6 inhibition induces an on-target feedback mechanism based on the activation of anti-apoptotic BH3 members. This oncogene-addition switching mechanism was harnessed to develop rational combinatorial therapies for GCB-DLBCL. PMID:26657288

  7. Sensory Rewiring in an Echolocator: Genome-Wide Modification of Retinogenic and Auditory Genes in the Bat Myotis davidii

    PubMed Central

    Hudson, Nicholas J.; Baker, Michelle L.; Hart, Nathan S.; Wynne, James W.; Gu, Quan; Huang, Zhiyong; Zhang, Guojie; Ingham, Aaron B.; Wang, Linfa; Reverter, Antonio

    2014-01-01

    Bats comprise 20% of all mammalian species and display a number of characteristics, including true flight, echolocation, and a heightened ability to resist viral load that uniquely position this group for comparative genomic studies. Here we searched for evidence of genomic variation consistent with sensory rewiring through bat evolution. We focused on two species with divergent sensory preferences. Myotis davidii is a bat species that echolocates and possesses dim- but not daylight-adapted vision whereas the black flying fox (Pteropus alecto) has highly developed day vision but does not echolocate. Using the naked mole rat as a reference, we found five functional genes (CYP1A2, RBP3, GUCY2F, CRYBB1, and GRK7) encoding visual proteins that have degenerated into pseudogenes in M. davidii but not P. alecto. In a second approach genome-wide codon usage bias (CUB) was compared between the two bat species. This CUB ranking systematically enriched for vision-related (CLN8, RD3, IKZF1, LAMC3, CRX, SOX8, VAX2, HPS1, RHO, PRPH2, and SOX9) and hearing-related (TPRN, TMIE, SLC52A3, OTOF, WFS1, SOD1, TBX18, MAP1A, OTOS, GPX1, and USH1G) machinery in M. davidii but not P. alecto. All vision and hearing genes selectively enriched in M. davidii for which orthologs could be identified also were more biased in the echolocating M. lucifugus than the nonecholocating P. vampyrus. We suggest that the existence of codon bias in vision- and hearing-related genes in a species that has evolved echolocation implies CUB is part of evolution’s toolkit to rewire sensory systems. We propose that the two genetic changes (pseudogene formation and CUB) collectively paint a picture of that incorporates a combination of destruction and gain-of-function. Together, they help explain how natural selection has reduced physiological costs associated with the development of a smaller eye poorly adapted to day vision but that also contribute to enhanced dim light vision and the hearing adaptations

  8. Sensory rewiring in an echolocator: genome-wide modification of retinogenic and auditory genes in the bat Myotis davidii.

    PubMed

    Hudson, Nicholas J; Baker, Michelle L; Hart, Nathan S; Wynne, James W; Gu, Quan; Huang, Zhiyong; Zhang, Guojie; Ingham, Aaron B; Wang, Linfa; Reverter, Antonio

    2014-10-01

    Bats comprise 20% of all mammalian species and display a number of characteristics, including true flight, echolocation, and a heightened ability to resist viral load that uniquely position this group for comparative genomic studies. Here we searched for evidence of genomic variation consistent with sensory rewiring through bat evolution. We focused on two species with divergent sensory preferences. Myotis davidii is a bat species that echolocates and possesses dim- but not daylight-adapted vision whereas the black flying fox (Pteropus alecto) has highly developed day vision but does not echolocate. Using the naked mole rat as a reference, we found five functional genes (CYP1A2, RBP3, GUCY2F, CRYBB1, and GRK7) encoding visual proteins that have degenerated into pseudogenes in M. davidii but not P. alecto. In a second approach genome-wide codon usage bias (CUB) was compared between the two bat species. This CUB ranking systematically enriched for vision-related (CLN8, RD3, IKZF1, LAMC3, CRX, SOX8, VAX2, HPS1, RHO, PRPH2, and SOX9) and hearing-related (TPRN, TMIE, SLC52A3, OTOF, WFS1, SOD1, TBX18, MAP1A, OTOS, GPX1, and USH1G) machinery in M. davidii but not P. alecto. All vision and hearing genes selectively enriched in M. davidii for which orthologs could be identified also were more biased in the echolocating M. lucifugus than the nonecholocating P. vampyrus. We suggest that the existence of codon bias in vision- and hearing-related genes in a species that has evolved echolocation implies CUB is part of evolution's toolkit to rewire sensory systems. We propose that the two genetic changes (pseudogene formation and CUB) collectively paint a picture of that incorporates a combination of destruction and gain-of-function. Together, they help explain how natural selection has reduced physiological costs associated with the development of a smaller eye poorly adapted to day vision but that also contribute to enhanced dim light vision and the hearing adaptations

  9. Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets

    PubMed Central

    Cass, Ashley A.; Bahn, Jae Hoon; Lee, Jae-Hyung; Greer, Christopher; Lin, Xianzhi; Kim, Yong; Hsiao, Yun-Hua Esther; Xiao, Xinshu

    2016-01-01

    In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation. PMID:26975654

  10. Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies.

    PubMed

    Dalmazzo, Leandro F F; Santana-Lemos, Bárbara A; Jácomo, Rafael H; Garcia, Aglair B; Rego, Eduardo M; da Fonseca, Luiz M; Falcão, Roberto P

    2011-05-01

    Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. PMID:21168913

  11. Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates.

    PubMed

    Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S; Schatz, David G

    2007-08-31

    The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. PMID:17613522

  12. Marinobufagenin-induced vascular fibrosis is a likely target for mineralocorticoid antagonists

    PubMed Central

    Fedorova, Olga V.; Emelianov, Igor V.; Bagrov, Konstantin A.; Grigorova, Yulia N.; Wei, Wen; Juhasz, Ondrej; Frolova, Elena V.; Marshall, Courtney A.; Lakatta, Edward G.; Konradi, Alexandra O.; Bagrov, Alexei Y.

    2015-01-01

    Objective Endogenous cardiotonic steroids (CTS), including marinobufagenin (MBG), stimulate vascular synthesis of collagen. Because mineralocorticoid antagonists competitively antagonize effect of CTS on the Na/K-ATPase, we hypothesized that spironolactone would reverse the pro-fibrotic effects of MBG. Methods Experiment 1. Explants of thoracic aortae and aortic vascular smooth muscle cells (VSMC) from Wistar rats were cultured for 24 hours in the presence of vehicle or MBG (100 nmol/L) with or without canrenone (10 µmol/L), an active metabolite of spironolactone. Experiment 2. In 16 patients (56 ± 2 yrs) with resistant hypertension (RH) on a combined (Lisinopril / amlodipine / hydrochlorothiazide) therapy, we determined arterial pressure, pulse wave velocity (PWV), plasma MBG, and erythrocyte Na/K-ATPase before and six months after addition of placebo (n=8) or spironolactone (50 mg/day; n=8) to the therapy. Results In rat aortic explants and in VSMC, pretreatment with MBG resulted in a two-fold rise in collagen-1, and a marked reduction in the sensitivity of the aortic rings to the vasorelaxant effect of sodium nitroprusside following endothelin-1-induced constriction (EC50=480±67 nmol/L vs. 23±3 nmol/L in vehicle-treated rings; P<0.01). Canrenone blocked effects of MBG on collagen synthesis and restored sensitivity of vascular rings to sodium nitroprusside (EC50 = 17±1 nmol/L). RH patients exhibited elevated plasma MBG (0.42 ± 0.07 vs. 0.24 ± 0.03 nmol/L; P=0.01) and reduced Na/K-ATPase activity (1.9 ± 0.15 vs 2.8 ± 0.2 µmol Pi/ml/hr, P<0.01) vs. 7 healthy subjects. Six-month administration of spironolactone, unlike placebo treatment, was associated with a decrease in PWV and arterial pressure, and with restoration of Na/K-ATPase activity in the presence of unchanged MBG levels. Conclusion MBG-induced vascular fibrosis is a likely target for spironolactone. PMID:26136067

  13. Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

    SciTech Connect

    Ragnum, Harald Bull; Røe, Kathrine; Holm, Ruth; Vlatkovic, Ljiljana; Nesland, Jahn Marthin; Aarnes, Eva-Katrine; Ree, Anne Hansen; Flatmark, Kjersti; Seierstad, Therese; Lilleby, Wolfgang; Lyng, Heidi

    2013-11-15

    Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to

  14. Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells.

    PubMed

    Zhao, Qingxia; Zhao, Ming; Parris, Amanda B; Xing, Ying; Yang, Xiaohe

    2016-09-01

    Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents. PMID:27574003

  15. Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells

    PubMed Central

    Zhao, Qingxia; Zhao, Ming; Parris, Amanda B.; Xing, Ying; Yang, Xiaohe

    2016-01-01

    Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents.

  16. Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs)*

    PubMed Central

    Ma, Ning; Shan, Yongli; Liao, Baojian; Kong, Guanyi; Wang, Cheng; Huang, Ke; Zhang, Hui; Cai, Xiujuan; Chen, Shubin; Pei, Duanqing; Chen, Nansheng; Pan, Guangjin

    2015-01-01

    The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. PMID:25795783

  17. Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling.

    PubMed

    Kang, Hee-Bum; Fan, Jun; Lin, Ruiting; Elf, Shannon; Ji, Quanjiang; Zhao, Liang; Jin, Lingtao; Seo, Jae Ho; Shan, Changliang; Arbiser, Jack L; Cohen, Cynthia; Brat, Daniel; Miziorko, Henry M; Kim, Eunhee; Abdel-Wahab, Omar; Merghoub, Taha; Fröhling, Stefan; Scholl, Claudia; Tamayo, Pablo; Barbie, David A; Zhou, Lu; Pollack, Brian P; Fisher, Kevin; Kudchadkar, Ragini R; Lawson, David H; Sica, Gabriel; Rossi, Michael; Lonial, Sagar; Khoury, Hanna J; Khuri, Fadlo R; Lee, Benjamin H; Boggon, Titus J; He, Chuan; Kang, Sumin; Chen, Jing

    2015-08-01

    Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development. PMID:26145173

  18. Key genes for modulating information flow play a temporal role as breast tumor coexpression networks are dynamically rewired by letrozole

    PubMed Central

    2013-01-01

    Background Genes do not act in isolation but instead as part of complex regulatory networks. To understand how breast tumors adapt to the presence of the drug letrozole, at the molecular level, it is necessary to consider how the expression levels of genes in these networks change relative to one another. Methods Using transcriptomic data generated from sequential tumor biopsy samples, taken at diagnosis, following 10-14 days and following 90 days of letrozole treatment, and a pairwise partial correlation statistic, we build temporal gene coexpression networks. We characterize the structure of each network and identify genes that hold prominent positions for maintaining network integrity and controlling information-flow. Results Letrozole treatment leads to extensive rewiring of the breast tumor coexpression network. Approximately 20% of gene-gene relationships are conserved over time in the presence of letrozole while 80% of relationships are condition dependent. The positions of influence within the networks are transiently held with few genes stably maintaining high centrality scores across the three time points. Conclusions Genes integral for maintaining network integrity and controlling information flow are dynamically changing as the breast tumor coexpression network adapts to perturbation by the drug letrozole. PMID:23819860

  19. Large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection.

    PubMed

    Li, Ze-Yuan; Xia, Jing; Chen, Zheng; Yu, Yang; Li, Quan-Feng; Zhang, Yu-Chan; Zhang, Jin-Ping; Wang, Cong-Ying; Zhu, Xiao-Yuan; Zhang, Weixiong; Chen, Yue-Qin

    2016-01-01

    Rice blast is a recurrent fungal disease, and resistance to fungal infection is a complex trait. Therefore, a comprehensive examination of rice transcriptome and its variation during fungal infection is necessary to understand the complex gene regulatory networks. In this study, adopting Next-Generation Sequencing we profiled the transcriptomes and microRNAomes of rice varieties, one susceptible and the other resistant to M. oryzae, at multiple time points during the fungal infection. Our results revealed a substantial variation in the plant transcriptome and microRNAome as well as change to rice innate immunity during fungal infection. A number of putative R gene candidates were identified from a perturbed rice transcriptome analysis. The expression of genes and non-coding RNA molecules changed in both fungal resistant and susceptible plants during M. oryzae invasion discovered distinct pathways triggered in the susceptible and resistant plants. In addition, a number of fungus genes in the susceptible and resistant plants were constantly expressed at different time points, suggesting that they were likely to be the potential AVR genes. Our results revealed large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection, which would help to develop more robust blast-resistant rice plants. PMID:27150822

  20. Large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection

    PubMed Central

    Li, Ze-Yuan; Xia, Jing; Chen, Zheng; Yu, Yang; Li, Quan-Feng; Zhang, Yu-Chan; Zhang, Jin-Ping; Wang, Cong-Ying; Zhu, Xiao-Yuan; Zhang, Weixiong; Chen, Yue-Qin

    2016-01-01

    Rice blast is a recurrent fungal disease, and resistance to fungal infection is a complex trait. Therefore, a comprehensive examination of rice transcriptome and its variation during fungal infection is necessary to understand the complex gene regulatory networks. In this study, adopting Next-Generation Sequencing we profiled the transcriptomes and microRNAomes of rice varieties, one susceptible and the other resistant to M. oryzae, at multiple time points during the fungal infection. Our results revealed a substantial variation in the plant transcriptome and microRNAome as well as change to rice innate immunity during fungal infection. A number of putative R gene candidates were identified from a perturbed rice transcriptome analysis. The expression of genes and non-coding RNA molecules changed in both fungal resistant and susceptible plants during M. oryzae invasion discovered distinct pathways triggered in the susceptible and resistant plants. In addition, a number of fungus genes in the susceptible and resistant plants were constantly expressed at different time points, suggesting that they were likely to be the potential AVR genes. Our results revealed large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection, which would help to develop more robust blast-resistant rice plants. PMID:27150822

  1. Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

    PubMed

    Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

    2015-09-01

    Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI. PMID:26301894

  2. Global Proteomics and Pathway Analysis of Pressure-overload Induced Heart Failure and Its Attenuation by Mitochondrial Targeted Peptides

    PubMed Central

    Dai, Dao-Fu; Hsieh, Edward J.; Chen, Tony; Menendez, Lorena G.; Basisty, Nathan B.; Tsai, Lauren; Beyer, Richard P.; Crispin, David A.; Shulman, Nicholas J.; Szeto, Hazel H.; Tian, Rong; MacCoss, Michael J.; Rabinovitch, Peter S.

    2013-01-01

    Background We investigated the protective effects of mitochondrial-targeted antioxidant and protective peptides, SS31 and SS20, on cardiac function, proteomic remodeling and signaling pathways. Methods and Results We applied an improved label-free shotgun proteomics approach to evaluate the global proteomics changes in transverse aortic constriction (TAC) induced heart failure, and the associated signaling pathway changes using Ingenuity Pathway Analysis (IPA). We found 538 proteins significantly changed after TAC, which mapped to 53 pathways. The top pathways were in the categories of actin cytoskeleton, mitochondrial function, intermediate metabolism, glycolysis / gluconeogenesis and citrate cycle. Concomitant treatment with SS31 ameliorated the congestive heart failure phenotypes and mitochondrial damage induced by TAC, in parallel with global attenuation of mitochondrial proteome changes, with an average of 84% protection of mitochondrial and 69% of non-mitochondrial protein changes. This included significant amelioration of All the IPA pathways noted above. SS20 had only modest effects on heart failure and this tracked with only partial attenuation of global proteomics changes; furthermore, while actin cytoskeleton pathways were significantly protected in SS20, mitochondrial and metabolic pathways essentially were not. Conclusions This study elucidates the signaling pathways significantly changed in pressure-overload induced heart failure. The global attenuation of TAC-induced proteomic alterations by the mitochondrial targeted peptide SS-31 suggests that perturbed mitochondrial function may be an upstream signal to many of pathway alterations in TAC and supports the potential clinical application of mitochondrial-targeted peptide drugs for the treatment heart failure. PMID:23935006

  3. Targeted Drug Delivery with Focused Ultrasound-Induced Blood-Brain Barrier Opening Using Acoustically-Activated Nanodroplets

    PubMed Central

    Chen, Cherry C.; Sheeran, Paul S.; Wu, Shih-Ying; Olumolade, Oluyemi O.; Dayton, Paul A.; Konofagou, Elisa E.

    2013-01-01

    Focused ultrasound (FUS) in the presence of systemically administered microbubbles has been shown to locally, transiently and reversibly increase the permeability of the blood-brain barrier (BBB), thus allowing targeted delivery of therapeutic agents in the brain for the treatment of central nervous system diseases. Currently, microbubbles are the only agents that have been used to facilitate the FUS-induced BBB opening. However, they are constrained within the intravascular space due to their micron-size diameters, limiting the delivery effect at or near the microvessels. In the present study, acoustically-activated nanodroplets were used as a new class of contrast agents to mediate FUS-induced BBB opening in order to study the feasibility of utilizing these nanoscale phase-shift particles for targeted drug delivery in the brain. Significant dextran delivery was achieved in the mouse hippocampus using nanodroplets at clinically relevant pressures. Passive cavitation detection was used in the attempt to establish a correlation between the amount of dextran delivered in the brain and the acoustic emission recorded during sonication. Conventional microbubbles with the same lipid shell composition and perfluorobutane core as the nanodroplets were also used to compare the efficiency of FUS-induced dextran delivery. It was found that nanodroplets had a higher BBB opening pressure threshold but a lower stable cavitation threshold than microbubbles, suggesting that contrast agent-dependent acoustic emission monitoring was needed. More homogeneous dextran delivery within the targeted hippocampus was achieved using nanodroplets without inducing inertial cavitation or compromising safety. Our results offered a new means of developing the FUS-induced BBB opening technology for potential extravascular targeted drug delivery in the brain, extending the potential drug delivery region beyond the cerebral vasculature. PMID:24096019

  4. In Vivo Space Radiation-Induced Non-Targeted Responses: Late Effects On Molecular Signaling In Mitochondria

    PubMed Central

    Jain, Mohit R.; Li, Min; Chen, Wei; Liu, Tong; de Toledo, Sonia M.; Pandey, Badri N.; Li, Hong; Rabin, Bernard M.; Azzam, Edouard I.

    2012-01-01

    The lack of clear knowledge about space radiation-induced biological effects has been singled out as the most important factor limiting the prediction of radiation risk associated with human space exploration. The expression of space radiation-induced non-targeted effects is thought to impact our understanding of the health risks associated with exposure to low fluences of particulate radiation encountered by astronauts during prolonged space travel. Following a brief review of radiation-induced bystander effects and the growing literature for the involvement of oxidative metabolism in their expression, we show novel data on the induction of in vivo non-targeted effects following exposure to 1100 MeV/nucleon titanium ions. Analyses of proteins by two-dimensional gel electrophoresis in non-targeted liver of cranially-irradiated Sprague Dawley rats revealed that the levels of key proteins involved in mitochondrial fatty acid metabolism are decreased. In contrast, those of proteins involved in various cellular defense mechanisms, including antioxidation, were increased. These data contribute to our understanding of the mechanisms underlying the biological responses to space radiation, and support the involvement of mitochondrial processes in the expression of radiation induced non-targeted effects. Significantly, they reveal the cross-talk between propagated stressful effects and induced adaptive responses. Together, with the accumulating data in the field, our results may help reduce the uncertainty in the assessment of the health risks to astronauts. They further demonstrate that ‘network analyses’ is an effective tool towards characterizing the signaling pathways that mediate the long-term biological effects of space radiation. PMID:21166651

  5. Neutron yield enhancement in laser-induced deuterium-deuterium fusion using a novel shaped target.

    PubMed

    Zhao, J R; Zhang, X P; Yuan, D W; Chen, L M; Li, Y T; Fu, C B; Rhee, Y J; Li, F; Zhu, B J; Li, Yan F; Liao, G Q; Zhang, K; Han, B; Liu, C; Huang, K; Ma, Y; Li, Yi F; Xiong, J; Huang, X G; Fu, S Z; Zhu, J Q; Zhao, G; Zhang, J

    2015-06-01

    Neutron yields have direct correlation with the energy of incident deuterons in experiments of laser deuterated target interaction [Roth et al., Phys. Rev. Lett. 110, 044802 (2013) and Higginson et al., Phys. Plasmas 18, 100703 (2011)], while deuterated plasma density is also an important parameter. Experiments at the Shenguang II laser facility have produced neutrons with energy of 2.45 MeV using d (d, n) He reaction. Deuterated foil target and K-shaped target were employed to study the influence of plasma density on neutron yields. Neutron yield generated by K-shaped target (nearly 10(6)) was two times higher than by foil target because the K-shaped target results in higher density plasma. Interferometry and multi hydro-dynamics simulation confirmed the importance of plasma density for enhancement of neutron yields. PMID:26133837

  6. Neutron yield enhancement in laser-induced deuterium-deuterium fusion using a novel shaped target

    SciTech Connect

    Zhao, J. R.; Chen, L. M. Li, Y. T.; Li, F.; Zhu, B. J.; Li, Yan. F.; Liao, G. Q.; Huang, K.; Ma, Y.; Li, Yi. F.; Zhang, X. P.; Fu, C. B.; Yuan, D. W.; Zhang, K.; Han, B.; Zhao, G.; Rhee, Y. J.; Liu, C.; Xiong, J.; Huang, X. G.; and others

    2015-06-15

    Neutron yields have direct correlation with the energy of incident deuterons in experiments of laser deuterated target interaction [Roth et al., Phys. Rev. Lett. 110, 044802 (2013) and Higginson et al., Phys. Plasmas 18, 100703 (2011)], while deuterated plasma density is also an important parameter. Experiments at the Shenguang II laser facility have produced neutrons with energy of 2.45 MeV using d (d, n) He reaction. Deuterated foil target and K-shaped target were employed to study the influence of plasma density on neutron yields. Neutron yield generated by K-shaped target (nearly 10{sup 6}) was two times higher than by foil target because the K-shaped target results in higher density plasma. Interferometry and multi hydro-dynamics simulation confirmed the importance of plasma density for enhancement of neutron yields.

  7. The DREAM complex mediates GIST cell quiescence and is a novel therapeutic target to enhance imatinib-induced apoptosis.

    PubMed

    Boichuk, Sergei; Parry, Joshua A; Makielski, Kathleen R; Litovchick, Larisa; Baron, Julianne L; Zewe, James P; Wozniak, Agnieszka; Mehalek, Keith R; Korzeniewski, Nina; Seneviratne, Danushka S; Schöffski, Patrick; Debiec-Rychter, Maria; DeCaprio, James A; Duensing, Anette

    2013-08-15

    Gastrointestinal stromal tumors (GIST) can be successfully treated with imatinib mesylate (Gleevec); however, complete remissions are rare and patients frequently achieve disease stabilization in the presence of residual tumor masses. The clinical observation that discontinuation of treatment can lead to tumor progression suggests that residual tumor cells are, in fact, quiescent and, therefore, able to re-enter the cell-division cycle. In line with this notion, we have previously shown that imatinib induces GIST cell quiescence in vitro through the APC(CDH1)-SKP2-p27(Kip1) signaling axis. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and that this process also involves the DREAM complex, a multisubunit complex that has recently been identified as an additional key regulator of quiescence. Importantly, inhibition of DREAM complex formation by depletion of the DREAM regulatory kinase DYRK1A or its target LIN52 was found to enhance imatinib-induced cell death. Our results show that imatinib induces apoptosis in a fraction of GIST cells while, at the same time, a subset of cells undergoes quiescence involving the DREAM complex. Inhibition of this process enhances imatinib-induced apoptosis, which opens the opportunity for future therapeutic interventions to target the DREAM complex for more efficient imatinib responses. PMID:23786773

  8. Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death

    PubMed Central

    Yin, Li; Kufe, Turner; Avigan, David

    2014-01-01

    The proteosome inhibitor bortezomib (BTZ) induces endoplasmic reticulum and oxidative stress in multiple myeloma (MM) cells. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in most MM cells, and targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is effective in inducing reactive oxygen species (ROS)-mediated MM cell death. The present results demonstrate that GO-203 and BTZ synergistically downregulate expression of the p53-inducible regulator of glycolysis and apoptosis (TIGAR), which promotes shunting of glucose-6-phosphate into the pentose phosphate pathway to generate reduced glutathione (GSH). In turn, GO-203 blocks BTZ-induced increases in GSH and results in synergistic increases in ROS and MM cell death. The results also demonstrate that GO-203 is effective against BTZ-resistant MM cells. We show that BTZ resistance is associated with BTZ-induced increases in TIGAR and GSH levels, and that GO-203 resensitizes BTZ-resistant cells to BTZ treatment by synergistically downregulating TIGAR and GSH. The GO-203/BTZ combination is thus highly effective in killing BTZ-resistant MM cells. These findings support a model in which targeting MUC1-C is synergistic with BTZ in suppressing TIGAR-mediated regulation of ROS levels and provide an experimental rationale for combining GO-203 with BTZ in certain settings of BTZ resistance. PMID:24632713

  9. Aberrant Restoration of Spines and their Synapses in l-DOPA-Induced Dyskinesia: Involvement of Corticostriatal but Not Thalamostriatal Synapses

    PubMed Central

    Zhang, Yiyue; Mendoza-Elias, Nasya; Rademacher, David J.; Tseng, Kuei Y.; Steece-Collier, Kathy

    2013-01-01

    We examined the structural plasticity of excitatory synapses from corticostriatal and thalamostriatal pathways and their postsynaptic targets in adult Sprague-Dawley rats to understand how these striatal circuits change in l-DOPA-induced dyskinesias (LIDs). We present here detailed electron and light microscopic analyses that provide new insight into the nature of the structural and synaptic remodeling of medium spiny neurons in response to LIDs. Numerous studies have implicated enhanced glutamate signaling and persistent long-term potentiation as central to the behavioral sensitization phenomenon of LIDs. Moreover, experience-dependent alterations in behavior are thought to involve structural modifications, specifically alterations in patterns of synaptic connectivity. Thus, we hypothesized that in the striatum of rats with LIDs, one of two major glutamatergic pathways would form new or altered contacts, especially onto the spines of medium spiny neuron (MSNs). Our data provide compelling evidence for a dramatic rewiring of the striatum of dyskinetic rats and that this rewiring involves corticostriatal but not thalamostriatal contacts onto MSNs. There is a dramatic increase in corticostriatal contacts onto spines and dendrites that appear to be directly linked to dyskinetic behaviors, since they were not seen in the striatum of animals that did not develop dyskinesia. There is also an aberrant increase in spines receiving more than one excitatory contact(i.e., multisynaptic spines) in the dyskinetic animals compared with the 6-hydroxydopamine-treated and control rats. Such alterations could substantially impair the ability of striatal neurons to gate cortically driven signals and contribute to the loss of bidirectional synaptic plasticity. PMID:23843533

  10. Effects of material properties on laser-induced bubble formation in absorbing liquids and on submerged targets

    NASA Astrophysics Data System (ADS)

    Shangguan, HanQun; Casperson, Lee W.; Shearin, Alan; Paisley, Dennis L.; Prahl, Scott A.

    1997-05-01

    Pulsed laser ablation of blood clots in a fluid-filled blood vessel is accomplished by an explosive evaporation process. The resulting vapor bubble rapidly expands and collapses to disrupt the thrombus (blood clot). The hydrodynamic pressures following the bubble expansion and collapse can also be used as a driving force to deliver clot-dissolving agents into thrombus for enhancement of laser thrombolysis. Thus, the laser-induced bubble formation plays an important role in the thrombus removal process. In this study the effects of material properties on laser-induced cavitation bubbles formed in liquids and on submerged targets have been visualized with a microsecond strobe or high speed framing camera.

  11. Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model

    PubMed Central

    2013-01-01

    Background Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12. Methods To test the inducibility of the p21 promoter, three reporter gene experimental models with green fluorescent protein (GFP) under the control of p21 promoter were established by gene electrotransfer of plasmid DNA: stably transfected cells, stably transfected tumors, and transiently transfected muscles. Induction of reporter gene expression after irradiation was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope in vivo. The antitumor effect of the plasmid encoding the p21 promoter driven interleukin 12 after radio-gene therapy was determined by tumor growth delay assay and by quantification of intratumoral and serum levels of interleukin 12 protein and intratumoral concentrations of interleukin 12 mRNA. Results Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter. Conclusions In this

  12. Simulation of residual activity in steel and copper targets induced by 950 MeV/nucleon uranium ions

    NASA Astrophysics Data System (ADS)

    Beskrovnaia, L.; Latysheva, L.; Paraipan, M.; Sobolevsky, N.; Timoshenko, G.

    2011-07-01

    Radioactive nuclides will be produced in structures and surrounding materials of the relativistic heavy ion collider NICA. Predicting radioactivity in the materials, air, cooling water, and ground is an important part of the technical project. Some Monte Carlo transport codes (FLUKA, GEANT4, SHIELD) can simulate the production of radionuclides induced by heavy ions. The preliminary verification of MC codes with experimental data is necessary before using them for radiation protection purposes. In the present work, comparisons of the experiment and GEANT4 and SHIELD simulations of induced activity in thick stainless steel and copper targets irradiated with 950 MeV/nucleon uranium ions are presented.

  13. E2F1 localizes predominantly to neuronal cytoplasm and fails to induce expression of its transcriptional targets in Human Immunodeficiency Virus-induced neuronal damage

    PubMed Central

    Wang, Ying; Shyam, Nikhil; Ting, Jenhao H.; Akay, Cagla; Lindl, Kathryn A.; Jordan-Sciutto, Kelly L.

    2010-01-01

    As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary post-mitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By qPCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19ARF) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19ARF, Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity. PMID:20580656

  14. Non-Targeted Effects Induced by Ionizing Radiation: Mechanisms and Potential Impact on Radiation Induced Health Effects

    SciTech Connect

    Morgan, William F.; Sowa, Marianne B.

    2015-01-01

    Not-targeted effects represent a paradigm shift from the "DNA centric" view that ionizing radiation only elicits biological effects and subsequent health consequences as a result of an energy deposition event in the cell nucleus. While this is likely true at higher radiation doses (> 1Gy), at low doses (< 100mGy) non-targeted effects associated with radiation exposure might play a significant role. Here definitions of non-targeted effects are presented, the potential mechanisms for the communication of signals and signaling networks from irradiated cells/tissues are proposed, and the various effects of this intra- and intercellular signaling are described. We conclude with speculation on how these observations might lead to and impact long-term human health outcomes.

  15. Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes

    PubMed Central

    Wang, Lin-Feng; Su, Su-Wen; Wang, Lei; Zhang, Guo-Qiang; Zhang, Rong; Niu, Yu-Jie; Guo, Yan-Su; Li, Chun-Yan; Jiang, Wen-Bo; Liu, Yi; Guo, Hui-Cai

    2015-01-01

    Oxidative stress plays an important role in doxorubicin (DOX)-induced cardiotoxicity. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor that orchestrates the antioxidant and cytoprotective responses to oxidative stress. In the present study, we tested whether tert-butylhydroquinone (tBHQ) could protect against DOX-induced cardiotoxicity in vivo and, if so, whether the protection was associated with the up-regulation of the Nrf2 pathway. The results showed that treatment with tBHQ significantly decreased the DOX-induced cardiac injury in wild-type mice. Moreover, tBHQ ameliorated the DOX-induced oxidative stress and apoptosis. Further studies suggested that tBHQ increased the nuclear accumulation of Nrf2 and the Nrf2-regulated gene expression, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxido-reductase-1 (NQO-1) expression. Knocking out Nrf2 in mice abolished the protective effect of tBHQ on the DOX-induced cardiotoxicity. These results indicate that tBHQ has a beneficial effect on DOX-induced cardiotoxicity, and this effect was associated with the enhanced expression of Nrf2 and its downstream antioxidant genes, HO-1 and NQO-1. PMID:26692920

  16. Inhibition of Lapatinib-induced Kinome Reprogramming in ERBB2-positive Breast Cancer by Targeting BET Family Bromodomains

    PubMed Central

    Stuhlmiller, Timothy J.; Miller, Samantha M.; Zawistowski, Jon S.; Nakamura, Kazuhiro; Beltran, Adriana S.; Duncan, James S.; Angus, Steven P.; Collins, Kyla A. L.; Granger, Deborah A.; Reuther, Rachel A.; Graves, Lee M.; Gomez, Shawn M.; Kuan, Pei-Fen; Parker, Joel S.; Chen, Xin; Sciaky, Noah; Carey, Lisa A.; Earp, H. Shelton; Jin, Jian; Johnson, Gary L.

    2015-01-01

    SUMMARY Therapeutics such as lapatinib that target ERBB2 often provide initial clinical benefit but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib and overcoming the dilemma of heterogeneity in the adaptive response. PMID:25865888

  17. VEGFA/VEGFR2-targeted therapies prevent the VEGFA-induced proliferation of regulatory T cells in cancer

    PubMed Central

    Terme, Magali; Tartour, Eric; Taieb, Julien

    2013-01-01

    Some of the anti-angiogenic agents currently used to treat solid malignancies have effects on tumor endothelial cells as well as on immune cells. We have recently demonstrated that targeting the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) signaling pathway reduces the proportion of regulatory T cells (Treg) in a mouse model of colorectal cancer (CRC) and in metastatic CRC patients as it inhibits tumor-induced Treg proliferation. PMID:24083078

  18. Targeting Pro-Apoptotic TRAIL Receptors Sensitizes HeLa Cervical Cancer Cells to Irradiation-Induced Apoptosis

    SciTech Connect

    Maduro, John H.; Vries, Elisabeth de; Meersma, Gert-Jan; Hougardy, Brigitte; Zee, Ate G.J. van der; Jong, Steven de

    2008-10-01

    Purpose: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line. Methods and Materials: Recombinant human TRAIL (rhTRAIL) and the agonistic antibodies against DR4 and DR5 were added to irradiated HeLa cells. The effect was evaluated with apoptosis and cytotoxicity assays and at the protein level. Membrane receptor expression was measured with flow cytometry. Small-interfering RNA against p53, DR4, and DR5 was used to investigate their function on the combined effect. Results: rhTRAIL and the agonistic DR4 and DR5 antibodies strongly enhanced 10-Gy-induced apoptosis. This extra effect was 22%, 23%, and 29% for rhTRAIL, DR4, and DR5, respectively. Irradiation increased p53 expression and increased the membrane expression of DR5 and DR4. p53 suppression, as well as small-interfering RNA against DR5, resulted in a significant downregulation of DR5 membrane expression but did not affect apoptosis induced by irradiation and rhTRAIL. After small-interfering RNA against DR4, rhTRAIL-induced apoptosis and the additive effect of irradiation on rhTRAIL-induced apoptosis were abrogated, implicating an important role for DR4 in apoptosis induced through irradiation in combination with rhTRAIL. Conclusion: Irradiation-induced apoptosis is strongly enhanced by targeting the pro-apoptotic TRAIL receptors DR4 or DR5. Irradiation results in a p53-dependent increase in DR5 membrane expression. The sensitizing effect of rhTRAIL on irradiation in the HeLa cell line is, however especially mediated through the DR4 receptor.

  19. DNA Vaccine that Targets Hemagglutinin to MHC Class II Molecules Rapidly Induces Antibody-Mediated Protection against Influenza

    PubMed Central

    Mjaaland, Siri; Roux, Kenneth H.; Fredriksen, Agnete Brunsvik

    2013-01-01

    New influenza A viruses with pandemic potential periodically emerge due to viral genomic reassortment. In the face of pandemic threats, production of conventional egg-based vaccines is time consuming and of limited capacity. We have developed in this study a novel DNA vaccine in which viral hemagglutinin (HA) is bivalently targeted to MHC class II (MHC II) molecules on APCs. Following DNA vaccination, transfected cells secreted vaccine proteins that bound MHC II on APCs and initiated adaptive immune responses. A single DNA immunization induced within 8 d protective levels of strain-specific Abs and also cross-reactive T cells. During the Mexican flu pandemic, a targeted DNA vaccine (HA from A/California/07/2009) was generated within 3 wk after the HA sequences were published online. These results suggest that MHC II–targeted DNA vaccines could play a role in situations of pandemic threats. The vaccine principle should be extendable to other infectious diseases. PMID:23956431

  20. Targeted pulmonary delivery of inducers of host macrophage autophagy as a potential host-directed chemotherapy of tuberculosis.

    PubMed

    Gupta, Anuradha; Misra, Amit; Deretic, Vojo

    2016-07-01

    One of the promising host-directed chemotherapeutic interventions in tuberculosis (TB) is based on inducing autophagy as an immune effector. Here we consider the strengths and weaknesses of potential autophagy-based pharmacological intervention. Using the existing drugs that induce autophagy is an option, but it has limitations given the broad role of autophagy in most cells, tissues, and organs. Thus, it may be desirable that the agent being used to modulate autophagy is applied in a targeted manner, e.g. delivered to affected tissues, with infected macrophages being an obvious choice. This review addresses the advantages and disadvantages of delivering drugs to induce autophagy in M. tuberculosis-infected macrophages. One option, already being tested in models, is to design particles for inhalation delivery to lung macrophages. The choice of drugs, drug release kinetics and intracellular residence times, non-target cell exposure and feasibility of use by patients is discussed. We term here this (still experimental) approach, of compartment-targeting, autophagy-based, host-directed therapy as "Track-II antituberculosis chemotherapy." PMID:26829287

  1. A dendritic cell targeted vaccine induces long-term HIV-specific immunity within the gastrointestinal tract.

    PubMed

    Ruane, D; Do, Y; Brane, L; Garg, A; Bozzacco, L; Kraus, T; Caskey, M; Salazar, A; Trumpheller, C; Mehandru, S

    2016-09-01

    Despite significant therapeutic advances for HIV-1 infected individuals, a preventative HIV-1 vaccine remains elusive. Studies focusing on early transmission events, including the observation that there is a profound loss of gastrointestinal (GI) CD4(+) T cells during acute HIV-1 infection, highlight the importance of inducing HIV-specific immunity within the gut. Here we report on the generation of cellular and humoral immune responses in the intestines by a mucosally administered, dendritic cell (DC) targeted vaccine. Our results show that nasally delivered α-CD205-p24 vaccine in combination with polyICLC, induced polyfunctional immune responses within naso-pulmonary lymphoid sites that disseminated widely to systemic and mucosal (GI tract and the vaginal epithelium) sites. Qualitatively, while α-CD205-p24 prime-boost immunization generated CD4(+) T-cell responses, heterologous prime-boost immunization with α-CD205-p24 and NYVAC gag-p24 generated high levels of HIV-specific CD4(+) and CD8(+) T cells within the GI tract. Finally, DC-targeting enhanced the amplitude and longevity of vaccine-induced immune responses in the GI tract. This is the first report of a nasally delivered, DC-targeted vaccine to generate HIV-specific immune responses in the GI tract and will potentially inform the design of preventative approaches against HIV-1 and other mucosal infections. PMID:26732678

  2. Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

    PubMed Central

    Zhao, Ling-Ling; Liu, Yuan-Fang; Peng, Li-Jun; Fei, Ai-Mei; Cui, Wen; Miao, Sheng-Chao; Hermine, Olivier; Gressin, Remy; Khochbin, Saadi; Chen, Sai-Juan; Wang, Jin; Mi, Jian-Qing

    2015-01-01

    Although most of the mantle cell lymphoma (MCL) patients initially responded well to bortezomib (BTZ), the dose-dependent toxicities have greatly limited the application of BTZ to MCL. To investigate the efficacy and mechanism of arsenic trioxide (ATO) with BTZ in inducing apoptosis of MCL cells, two MCL cell lines, along with primary cells from MCL patients (n = 4), were used. Additionally, the NOD-SCID mice xenograft model of Jeko-1 cells was established to study the anti-MCL mechanisms in an in vivo setting. ATO treatment highly improved BTZ capacity to inhibit proliferation and induce apoptosis of MCL cells. Furthermore, the interaction of Noxa and Mcl-1 leads Bak to release from Mcl-1 or from Bcl-xl, which could further activate Bak and Bax and then induce cell apoptosis. We also found that when lower doses of BTZ were used in combination with ATO, more effective proapoptotic effects in both the cell lines and the primary cells were obtained compared to the effects of BTZ used alone at higher doses. Simultaneously, the combination of these two drugs delayed the tumor growth in mice more effectively than BTZ alone. The cooperative anti-MCL effects of this combination therapy both in vitro and in vivo strongly provided a new strategy to the clinical treatment of MCL. PMID:26310857

  3. Lipocalin 2, a new GADD153 target gene, as an apoptosis inducer of endoplasmic reticulum stress in lung cancer cells

    SciTech Connect

    Hsin, I-Lun; Hsiao, Yueh-Chieh; Wu, Ming-Fang; Jan, Ming-Shiou; Tang, Sheau-Chung; Lin, Yu-Wen; Hsu, Chung-Ping; Ko, Jiunn-Liang

    2012-09-15

    Endoplasmic reticulum (ER) stress is activated under severe cellular conditions. GADD153, a member of the C/EBP family, is an unfolded protein response (UPR) responsive transcription factor. Increased levels of lipocalin 2, an acute phase protein, have been found in several epithelial cancers. The aim of this study is to investigate the function of lipocalin 2 in lung cancer cells under ER stress. Treatment with thapsigargin, an ER stress activator, led to increases in cytotoxicity, ER stress, apoptosis, and lipocalin 2 expression in A549 cells. GADD153 silencing decreased lipocalin 2 expression in A549 cells. On chromatin immunoprecipitation assay, ER stress increased GADD153 DNA binding to lipocalin 2 promoter. Furthermore, silencing of lipocalin 2 mitigated ER stress-mediated apoptosis in A549 cells. Our findings demonstrated that lipocalin 2 is a new GADD153 target gene that mediates ER stress-induced apoptosis. Highlights: ► We demonstrate that Lipocalin 2 is a new GADD153 target gene. ► Lipocalin 2 mediates ER stress-induced apoptosis. ► ER stress-induced lipocalin 2 expression is calcium-independent in A549 cells. ► Lipocalin 2 dose not play a major role in ER stress-induced autophagy.

  4. Kamebakaurin inhibits the expression of hypoxia-inducible factor-1α and its target genes to confer antitumor activity.

    PubMed

    Wang, Ke Si; Ma, Juan; Mi, Chunliu; Li, Jing; Lee, Jung Joon; Jin, Xuejun

    2016-04-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Kamebakaurin is a diterpenoid compound isolated from Isodon excia (Maxin.) Hara, which has been used for anti-inflammatory activities. However, its antitumor activity along with molecular mechanism has not been reported. Kamebakaurin showed potent inhibitory activity against HIF-1 activation induced by hypoxia or CoCl2 in various human cancer cell lines. This compound significantly decreased the hypoxia-induced accumulation of HIF-1α protein, whereas it did not affect the expression of topoisomerase-I (Topo-I). Further analysis revealed that kamebakaurin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Furthermore, kamebakaurin prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin (EPO). However, kamebakaurin caused cell growth inhibition via cell cycle arrest at G1 phase in tumor cells. In vivo studies, we further confirmed the inhibitory effect of kamebakaurin on the expression of HIF-1α proteins, leading to growth inhibition of HCT116 cells in a xenograft tumor model. These results show that kamebakaurin is an effective inhibitor of HIF-1 and provide new perspectives into its anticancer activity. PMID:26781327

  5. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    SciTech Connect

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  6. miR-98 protects endothelial cells against hypoxia/reoxygenation induced-apoptosis by targeting caspase-3.

    PubMed

    Li, He-wen; Meng, Yan; Xie, Qun; Yi, Wen-jing; Lai, Xue-li; Bian, Qi; Wang, Jun; Wang, Jia-feng; Yu, Guang

    2015-11-20

    Endothelial dysfunction is one of the main pathophysiological processes involved in renal ischemia reperfusion injury. Our previous microarray study demonstrated that miR-98 was upregulated in the kidney with ischemia reperfusion injury (IRI). The present study was performed to investigate whether miR-98 was involved in the regulation of endothelial apoptosis under hypoxia and re-oxygenation (H/R) conditions. The dynamic changes of miR-98 in mouse IRI kidney and H/R HUVECs was measured. HUVECs were treated with HIF-1α siRNA to investigate the role of HIF-1α on miR-98 expression. The potential target genes of miR-98 were predicted by bioinformatics analyses. HUVECs were transfected with miR-98 mimics or inhibitor to confirm the role of miR-98 on the expression of target genes and hypoxia-induced apoptosis. The target gene was finally confirmed by dual-luciferase reporter assay. Both of IRI and H/R induced significantly up-regulation of miR-98 in the ischemic kidney and hypoxic HUVECs. HIF-1α siRNA remarkably down-regulated the expression of miR-98 in both normal and hypoxic HUVECs. The putative target genes of miR-98 included IL-6, IL-10 and caspase-3. MiR-98 mimics significantly inhibit caspase-3 expression in HUVECs, while anti-miR-98 significantly up-regulated it. But no change of IL-6 and IL-10 levels was observed after miRNA transfection. miR-98 protected HUVECs against apoptosis induced by hypoxia, while anti-miR-98 had the reverse effect. Furthermore, the dual-luciferase reporter assay confirmed that miR-98 decreased the luciferase activity by targeting the 3' untranslated region of caspase-3. In conclusion, Renal IRI induces up-regulation of miR-98 dependent on HIF-1α, which protects endothelial cells against apoptosis by targeting caspase-3. PMID:26367177

  7. Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity.

    PubMed

    Pogue, Sarah L; Taura, Tetsuya; Bi, Mingying; Yun, Yong; Sho, Angela; Mikesell, Glen; Behrens, Collette; Sokolovsky, Maya; Hallak, Hussein; Rosenstock, Moti; Sanchez, Eric; Chen, Haiming; Berenson, James; Doyle, Anthony; Nock, Steffen; Wilson, David S

    2016-01-01

    Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα's TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity. PMID:27611189

  8. Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

    NASA Astrophysics Data System (ADS)

    Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

    2014-08-01

    Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

  9. EGF induces microRNAs that target suppressors of cell migration: miR-15b targets MTSS1 in breast cancer.

    PubMed

    Kedmi, Merav; Ben-Chetrit, Nir; Körner, Cindy; Mancini, Maicol; Ben-Moshe, Noa Bossel; Lauriola, Mattia; Lavi, Sara; Biagioni, Francesca; Carvalho, Silvia; Cohen-Dvashi, Hadas; Schmitt, Fernando; Wiemann, Stefan; Blandino, Giovanni; Yarden, Yosef

    2015-03-17

    Growth factors promote tumor growth and metastasis. We found that epidermal growth factor (EGF) induced a set of 22 microRNAs (miRNAs) before promoting the migration of mammary cells. These miRNAs were more abundant in human breast tumors relative to the surrounding tissue, and their abundance varied among breast cancer subtypes. One of these miRNAs, miR-15b, targeted the 3' untranslated region of MTSS1 (metastasis suppressor protein 1). Although xenografts in which MTSS1 was knocked down grew more slowly in mice initially, longer-term growth was unaffected. Knocking down MTSS1 increased migration and Matrigel invasion of nontransformed mammary epithelial cells. Overexpressing MTSS1 in an invasive cell line decreased cell migration and invasiveness, decreased the formation of invadopodia and actin stress fibers, and increased the formation of cellular junctions. In tissues from breast cancer patients with the aggressive basal subtype, an inverse correlation occurred with the high expression of miRNA-15b and the low expression of MTSS1. Furthermore, low abundance of MTSS1 correlated with poor patient prognosis. Thus, growth factor-inducible miRNAs mediate mechanisms underlying the progression of cancer. PMID:25783158

  10. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    SciTech Connect

    Pan, Jian-Bo; Ji, Nan; Pan, Wen; Hong, Ru; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified.

  11. Dihydrobetulinic Acid Induces Apoptosis in Leishmania donovani by Targeting DNA Topoisomerase I and II: Implications in Antileishmanial Therapy

    PubMed Central

    Chowdhury, Arnab Roy; Mandal, Suparna; Goswami, Anindya; Ghosh, Monidipa; Mandal, Labanya; Chakraborty, Debabani; Ganguly, Agneyo; Tripathi, Gayatri; Mukhopadhyay, Sibabrata; Bandyopadhyay, Santu; Majumder, Hemanta K

    2003-01-01

    Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 μM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs. PMID:12765337

  12. Cross sections of α-induced reactions for targets with masses A ≈ 20-50 at low energies

    NASA Astrophysics Data System (ADS)

    Mohr, Peter

    2015-05-01

    A simple reduction scheme using so-called reduced energies and reduced cross sections allows the comparison of heavy-ion-induced reaction cross sections for a broad range of masses of projectile and target and over a wide energy range. A global behavior has been found for strongly bound projectiles whereas much larger reduced cross sections have been observed for weakly bound and halo projectiles. It has been shown that this simple reduction scheme works also well for -particle-induced reactions on heavy target nuclei, but very recently significant deviations have been seen for + 33S and + 23Na. Motivated by these unexpected discrepancies, the present study analyses -induced reaction cross sections for targets with masses 20-50. The study shows that the experimental data for -induced reactions on nuclei with 20-50 deviate slightly from the global behavior of reduced cross sections. However, in general the deviations evolve smoothly towards lower masses. The only significant outliers are the recent data for 33S and 23Na which are far above the general systematics, and some very old data may indicate that 36Ar and 40Ar are below the general trend. As expected, also the doubly magic 40Ca nucleus lies slightly below the results for its neighboring nuclei. Overall, the experimental data are nicely reproduced by a statistical model calculation utilizing the simple -nucleus potential by McFadden and Satchler. Simultaneously with the deviation of reduced cross sections from the general behavior, the outliers 23Na, 33S, 36Ar, and 40Ar also show significant disagreement between experiment and statistical model calculation.

  13. Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis

    PubMed Central

    Hudson, Matthew B.; Smuder, Ashley J.; Nelson, W. Bradley; Wiggs, Michael P.; Shimkus, Kevin L.; Fluckey, James D.; Szeto, Hazel H.; Powers, Scott K.

    2015-01-01

    Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV. PMID:26361212

  14. Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells

    PubMed Central

    2012-01-01

    Background Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo. Results In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition. Conclusion These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer. PMID:22913813

  15. Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling.

    PubMed

    Zhao, Yun-peng; Wei, Jian-lu; Tian, Qing-yun; Liu, Alexander Tianxing; Yi, Young-su; Einhorn, Thomas A; Liu, Chuan-ju

    2016-01-01

    Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFα. Here we report that titanium particles significantly induced PGRN expression in RAW264.7 cells and also in a mouse air-pouch model of inflammation. PGRN-deficiency enhanced, whereas administration of recombinant PGRN effectively inhibited, titanium particle-induced inflammation in an air pouch model. In addition, PGRN also significantly inhibited titanium particle-induced osteoclastogenesis and calvarial osteolysis in vitro, ex vivo and in vivo. Mechanistic studies demonstrated that the inhibition of PGRN on titanium particle induced-inflammation is primarily via neutralizing the titanium particle-activated TNFα/NF-κB signaling pathway and this is evidenced by the suppression of particle-induced IκB phosphorylation, NF-κB p65 nuclear translocation, and activity of the NF-κB-specific reporter gene. Collectively, these findings not only demonstrate that PGRN plays an important role in inhibiting titanium particle-induced inflammation, but also provide a potential therapeutic agent for the prevention of wear debris-induced inflammation and osteolysis. PMID:26864916

  16. Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling

    PubMed Central

    Zhao, Yun-peng; Wei, Jian-lu; Tian, Qing-yun; Liu, Alexander Tianxing; Yi, Young-Su; Einhorn, Thomas A.; Liu, Chuan-ju

    2016-01-01

    Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFα. Here we report that titanium particles significantly induced PGRN expression in RAW264.7 cells and also in a mouse air-pouch model of inflammation. PGRN-deficiency enhanced, whereas administration of recombinant PGRN effectively inhibited, titanium particle-induced inflammation in an air pouch model. In addition, PGRN also significantly inhibited titanium particle-induced osteoclastogenesis and calvarial osteolysis in vitro, ex vivo and in vivo. Mechanistic studies demonstrated that the inhibition of PGRN on titanium particle induced-inflammation is primarily via neutralizing the titanium particle-activated TNFα/NF-κB signaling pathway and this is evidenced by the suppression of particle-induced IκB phosphorylation, NF-κB p65 nuclear translocation, and activity of the NF-κB-specific reporter gene. Collectively, these findings not only demonstrate that PGRN plays an important role in inhibiting titanium particle-induced inflammation, but also provide a potential therapeutic agent for the prevention of wear debris-induced inflammation and osteolysis. PMID:26864916

  17. Specific markers, micro-environmental anomalies and tropism: opportunities for gold nanorods targeting of tumors in laser-induced hyperthermia

    NASA Astrophysics Data System (ADS)

    Tatini, Francesca; Ratto, Fulvio; Centi, Sonia; Landini, Ida; Nobili, Stefania; Witort, Ewa; Fusi, Franco; Capaccioli, Sergio; Mini, Enrico; Pini, Roberto

    2014-03-01

    Gold nanorods (GNRs) are optimal contrast agents for near-infrared (NIR) laser-induced photothermal ablation of cancer. Selective targeting of cancer cells can be pursued by attaching specific molecules on the particles surface or by the use of cellular vectors loaded with GNRs. We performed and tested various targeting approaches by means of GNRs functionalization with (i) antibodies against Cancer-Antigen-125 (CA-125), (ii) inhibitors of the carbonic anhydrase 9 (CA9) and (iii) by the use of macrophages as cellular vectors. GNRs with a NIR absorption band at 810 nm were synthesized and PEGylated. For GNRs functionalization the targets of choice were CA-125, the most widely used biomarker for ovarian cancer, and CA9, overexpressed by hypoxic cells which are often located within the tumor mass. In the case of cellular vectors, to be used as Trojan horses naturally able to reach tumor areas, the surface of PEG-GNRs was modified to achieve unspecific interactions with macrophage membranes. In all cases the cellular uptake was evaluated by silver staining and cell viability was assessed by MTT test. Then tests of laser-induced GNRs-mediated hyperthermia were performed in various cell cultures illuminating with an 810 nm diode laser (CW, 0,5-4 W/cm2 power density, 1-10 min exposure time) and cell death was evaluated. Each targeting strategy we tested may be used alone or in combination, to maximize the tumor loading and therefore the efficiency of the laser treatment. Moreover, a multiple approach could help when the tumor variability interferes with the targeting directed to a single marker.

  18. Conditional Epistatic Interaction Maps Reveal Global Functional Rewiring of Genome Integrity Pathways in Escherichia coli.

    PubMed

    Kumar, Ashwani; Beloglazova, Natalia; Bundalovic-Torma, Cedoljub; Phanse, Sadhna; Deineko, Viktor; Gagarinova, Alla; Musso, Gabriel; Vlasblom, James; Lemak, Sofia; Hooshyar, Mohsen; Minic, Zoran; Wagih, Omar; Mosca, Roberto; Aloy, Patrick; Golshani, Ashkan; Parkinson, John; Emili, Andrew; Yakunin, Alexander F; Babu, Mohan

    2016-01-26

    As antibiotic resistance is increasingly becoming a public health concern, an improved understanding of the bacterial DNA damage response (DDR), which is commonly targeted by antibiotics, could be of tremendous therapeutic value. Although the genetic components of the bacterial DDR have been studied extensively in isolation, how the underlying biological pathways interact functionally remains unclear. Here, we address this by performing systematic, unbiased, quantitative synthetic genetic interaction (GI) screens and uncover widespread changes in the GI network of the entire genomic integrity apparatus of Escherichia coli under standard and DNA-damaging growth conditions. The GI patterns of untreated cultures implicated two previously uncharacterized proteins (YhbQ and YqgF) as nucleases, whereas reorganization of the GI network after DNA damage revealed DDR roles for both annotated and uncharacterized genes. Analyses of pan-bacterial conservation patterns suggest that DDR mechanisms and functional relationships are near universal, highlighting a modular and highly adaptive genomic stress response. PMID:26774489

  19. Potential of Inducible Nitric Oxide Synthase as a Therapeutic Target for Allergen-Induced Airway Hyperresponsiveness: A Critical Connection to Nitric Oxide Levels and PARP Activity.

    PubMed

    Ibba, Salome' V; Ghonim, Mohamed A; Pyakurel, Kusma; Lammi, Matthew R; Mishra, Anil; Boulares, A Hamid

    2016-01-01

    Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma. PMID:27524861

  20. Potential of Inducible Nitric Oxide Synthase as a Therapeutic Target for Allergen-Induced Airway Hyperresponsiveness: A Critical Connection to Nitric Oxide Levels and PARP Activity

    PubMed Central

    Ghonim, Mohamed A.; Pyakurel, Kusma; Mishra, Anil

    2016-01-01

    Although expression of inducible NO synthase (iNOS) in the lungs of asthmatics and associated nitrosative damage are established, iNOS failed as a therapeutic target for blocking airway hyperresponsiveness (AHR) and inflammation in asthmatics. This dichotomy calls for better strategies with which the enzyme is adequately targeted. Here, we confirm iNOS expression in the asthmatic lung with concomitant protein nitration and poly(ADP-ribose) polymerase (PARP) activation. We show, for the first time, that iNOS is highly expressed in peripheral blood mononuclear cells (PBMCs) of asthmatics with uncontrolled disease, which did not correspond to protein nitration. Selective iNOS inhibition with L-NIL protected against AHR upon acute, but not chronic, exposure to ovalbumin or house dust mite (HDM) in mice. Supplementation of NO by nitrite administration significantly blocked AHR in chronically HDM-exposed mice that were treated with L-NIL. Protection against chronic HDM exposure-induced AHR by olaparib-mediated PARP inhibition may be associated with the partial but not the complete blockade of iNOS expression. Indeed, L-NIL administration prevented olaparib-mediated protection against AHR in chronically HDM-exposed mice. Our study suggests that the amount of iNOS and NO are critical determinants in the modulation of AHR by selective iNOS inhibitors and renews the potential of iNOS as a therapeutic target for asthma. PMID:27524861

  1. Laser ablation of a platinum target in water. III. Laser-induced reactions

    SciTech Connect

    Nichols, William T.; Sasaki, Takeshi; Koshizaki, Naoto

    2006-12-01

    This is the third paper in our series studying the laser-target-liquid interactions occurring in laser ablation in liquids (LAL). Here, laser ablation of a platinum target in pure water at 355 nm wavelength is studied as a function of laser energy. We describe three distinct reaction regimes between the ablated target species and water at different laser focusing conditions. At low laser fluence (<10 J/cm{sup 2}), material removal is caused by laser heating of the platinum surface and the primary products are small clusters with a large percentage of platinum atoms in a nonzero oxidation state. At intermediate fluences (10-70 J/cm{sup 2}), platinum nanoparticles are the primary products. Our previous studies demonstrated that in this fluence regime ablation occurs through both thermal vaporization and explosive ejection of molten droplets. In both cases reactivity is small due to the low reactivity of platinum with water. At high fluences (>70 J/cm{sup 2}), we find large, faceted particles that are attributed to the drying of PtO{sub x} gels formed by reactive plasma etching of the target. Taken together these results demonstrate that significant tunability in the target-liquid interaction is possible during nanomaterial synthesis by LAL.

  2. NADPH oxidases are critical targets for prevention of ethanol-induced bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms through which chronic alcohol consumption induce bone loss and osteoporosis are largely unknown. Ethanol increases expression and activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzymes (Nox) in osteoblasts leading to accumulation of reactive oxygen spe...

  3. NOVEL MOLECULAR TARGETS IMPLICATED IN TESTICULAR DYSGENESIS INDUCED BY GESTATIONAL EXPOSURE TO DIETHYLHEXYL PHTHALATE (DEHP)

    EPA Science Inventory

    Phthalate-induced Testicular Dysgenesis Syndrome describes reproductive alterations in human males such as: hypospadias, cryptorchism, low sperm counts, and testicular cancer. This work is the first comprehensive evaluation of the rat fetal testis proteome following phthalate exp...

  4. RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

    PubMed Central

    Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

    2015-01-01

    MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

  5. Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy

    PubMed Central

    Wilfinger, Nastasia; Austin, Shane; Scheiber-Mojdehkar, Barbara; Berger, Walter; Reipert, Siegfried; Praschberger, Monika; Paur, Jakob; Trondl, Robert; Keppler, Bernhard K.; Zielinski, Christoph C.; Nowikovsky, Karin

    2016-01-01

    This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway. PMID:26517689

  6. MiR-708 promotes steroid-induced osteonecrosis of femoral head, suppresses osteogenic differentiation by targeting SMAD3

    PubMed Central

    Hao, Cheng; Yang, Shuhua; Xu, Weihua; Shen, Jacson K.; Ye, Shunan; Liu, Xianzhe; Dong, Zhe; Xiao, Baojun; Feng, Yong

    2016-01-01

    Steroid-induced osteonecrosis of femoral head (ONFH) is a serious complication of glucocorticoid (GC) use. We investigated the differential expression of miRs in the mesenchymal stem cells (MSCs) of patients with ONFH, and aimed to explain the relationship between GC use and the development of MSC dysfunction in ONFH. Cells were collected from bone marrow of patients with ONFH. Samples were assigned to either GCs Group or Control Group at 1:1 matched with control. We then used miRNA microarray analysis and real-time PCR to identify the differentially expressed miRs. We also induced normal MSCs with GCs to verify the differential expression above. Subsequently, we selected some of the miRs for further studies, including miRNA target and pathway prediction, and functional analysis. We discovered that miR-708 was upregulated in ONFH patients and GC-treated MSCs. SMAD3 was identified as a direct target gene of miR-708, and functional analysis demonstrated that miR-708 could markedly suppress osteogenic differentiation and adipogenesis differentiation of MSCs. Inhibition of miR-708 rescued the suppressive effect of GC on osteonecrosis. Therefore, we determined that GC use resulted in overexpression of miR-708 in MSCs, and thus, targeting miR-708 may serve as a novel therapeutic biomarker for the prevention and treatment of ONFH. PMID:26932538

  7. Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity.

    PubMed

    Yan, Haoheng; Endo, Yukinori; Shen, Yi; Rotstein, David; Dokmanovic, Milos; Mohan, Nishant; Mukhopadhyay, Partha; Gao, Bin; Pacher, Pal; Wu, Wen Jin

    2016-03-01

    Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs. PMID:26712117

  8. Glutathione prevents preterm parturition and fetal death by targeting macrophage-induced reactive oxygen species production in the myometrium.

    PubMed

    Hadi, Tarik; Bardou, Marc; Mace, Guillaume; Sicard, Pierre; Wendremaire, Maeva; Barrichon, Marina; Richaud, Sarah; Demidov, Oleg; Sagot, Paul; Garrido, Carmen; Lirussi, Frédéric

    2015-06-01

    Preterm birth is an inflammatory process resulting from the massive infiltration of innate immune cells and the production of proinflammatory cytokines in the myometrium. However, proinflammatory cytokines, which induce labor in vivo, fail to induce labor-associated features in human myometrial cells (MCs). We thus aimed to investigate if reactive oxygen species (ROS) production could be the missing step between immune cell activation and MC response. Indeed, we found that ROS production is increased in the human preterm laboring myometrium (27% ROS producing cells, respectively, versus 2% in nonlaboring controls), with 90% ROS production in macrophages. Using LPS-stimulated myometrial samples and cell coculture experiments, we demonstrated that ROS production is required for labor onset. Furthermore, we showed that ROS are required first in the NADPH oxidase (NADPHox)-2/NF-κB-dependent macrophage response to inflammatory stimuli but, more importantly, to trigger macrophage-induced MCs transactivation. Remarkably, in a murine model of LPS-induced preterm labor (inducing delivery within 17 hours, with no pup survival), cotreatment with glutathione delayed labor onset up to 94 hours and prevented in utero fetal distress, allowing 46% pups to survive. These results suggest that targeting ROS production with the macrophage-permeable antioxidant glutathione could constitute a promising strategy to prevent preterm birth. PMID:25757563

  9. Stress-Induced Pain: A Target for the Development of Novel Therapeutics

    PubMed Central

    Johnson, Anthony C.

    2014-01-01

    Although current therapeutics provide relief from acute pain, drugs used for treatment of chronic pain are typically less efficacious and limited by adverse side effects, including tolerance, addiction, and gastrointestinal upset. Thus, there is a significant need for novel therapies for the treatment of chronic pain. In concert with chronic pain, persistent stress facilitates pain perception and sensitizes pain pathways, leading to a feed-forward cycle promoting chronic pain disorders. Stress exacerbation of chronic pain suggests that centrally acting drugs targeting the pain- and stress-responsive brain regions represent a valid target for the development of novel therapeutics. This review provides an overview of how stress modulates spinal and central pain pathways, identifies key neurotransmitters and receptors within these pathways, and highlights their potential as novel targets for therapeutics to treat chronic pain. PMID:25194019

  10. High energy irradiations simulating cosmic-ray-induced planetary gamma ray production. I - Fe target

    NASA Technical Reports Server (NTRS)

    Metzger, A. E.; Parker, R. H.; Yellin, J.

    1986-01-01

    Two thick Fe targets were bombarded by a series of 6 GeV proton irradiations for the purpose of simulating the cosmic ray bombardment of planetary objects in space. Gamma ray energy spectra were obtained with a germanium solid state detector during the bombardment, and 46 of the gamma ray lines were ascribed to the Fe targets. A comparison between observed and predicted values showed good agreement for Fe lines from neutron inelastic scattering and spallation reactions, and less satisfactory agreement for neutron capture reactions, the latter attributed to the difference in composition between the Fe target and the mean lunar abundance used in the modeling. Through an analysis of the irradiation results together with continuum data obtained in lunar orbit, it was found that 100 hours of measurement with a current instrument should generate a spectrum containing approximately 20 lines due to Fe alone, with a 2-sigma sensitivity for detection of about 0.2 percent.

  11. A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification.

    PubMed

    Ma, Cuiping; Wang, Wenshuo; Mulchandani, Ashok; Shi, Chao

    2014-07-15

    A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs. PMID:24780220

  12. Prostate Specific Membrane Antigen-Targeted Photodynamic Therapy Induces Rapid Cytoskeletal Disruption

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Berkman, Clifford E.

    2010-01-01

    Prostate-specific membrane antigen (PSMA), an established enzyme-biomarker for prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications. We aimed to determine the effects of PSMA-targeted photodynamic therapy (PDT) on cytoskeletal networks in prostate cancer cells. PSMA-targeted PDT resulted in rapid disruption of microtubules (α-/β-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm of LNCaP cells. The collapse of cytoplasmic microtubules and the later nuclear translocation of α-/β-tubulin were the most dramatic alternation. It is likely that these early changes of cytoskeletal networks are partly involved in the initiation of cell death. PMID:20452720

  13. Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition.

    PubMed

    Ogasawara, Daisuke; Deng, Hui; Viader, Andreu; Baggelaar, Marc P; Breman, Arjen; den Dulk, Hans; van den Nieuwendijk, Adriann M C H; Soethoudt, Marjolein; van der Wel, Tom; Zhou, Juan; Overkleeft, Herman S; Sanchez-Alavez, Manuel; Mo, Simone; Nguyen, William; Conti, Bruno; Liu, Xiaojie; Chen, Yao; Liu, Qing-Song; Cravatt, Benjamin F; van der Stelt, Mario

    2016-01-01

    Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network. PMID:26668358

  14. Nonlinear surface plasma wave induced target normal sheath acceleration of protons

    SciTech Connect

    Liu, C. S.; Tripathi, V. K. Shao, Xi; Liu, T. C.

    2015-02-15

    The mode structure of a large amplitude surface plasma wave (SPW) over a vacuum–plasma interface, including relativistic and ponderomotive nonlinearities, is deduced. It is shown that the SPW excited by a p-polarized laser on a rippled thin foil target can have larger amplitude than the transmitted laser amplitude and cause stronger target normal sheath acceleration of protons as reported in a recent experiment. Substantial enhancement in proton number also occurs due to the larger surface area covered by the SPW.

  15. STAT3 as a target for inducing apoptosis in solid and hematological tumors

    PubMed Central

    Siddiquee, Khandaker Al Zaid; Turkson, James

    2008-01-01

    Studies in the past few years have provided compelling evidence for the critical role of aberrant Signal Transducer and Activator of Transcription 3 (STAT3) in malignant transformation and tumorigenesis. Thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design. This review focuses on aberrant STAT3 and its role in promoting tumor cell survival and supporting the malignant phenotype. A brief evaluation of the current strategies targeting STAT3 for the development of novel anticancer agents against human tumors harboring constitutively active STAT3 will also be presented. PMID:18227858

  16. Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

    PubMed

    Formey, Damien; Martín-Rodríguez, José Ángel; Leija, Alfonso; Santana, Olivia; Quinto, Carmen; Cárdenas, Luis; Hernández, Georgina

    2016-01-01

    A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean-rhizobia symbiosis. PMID:27271618

  17. Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells

    PubMed Central

    Formey, Damien; Martín-Rodríguez, José Ángel; Leija, Alfonso; Santana, Olivia; Quinto, Carmen; Cárdenas, Luis; Hernández, Georgina

    2016-01-01

    A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean–rhizobia symbiosis. PMID:27271618

  18. MicroRNA-7 Protects against 1-Methyl-4-Phenylpyridinium-Induced Cell Death by Targeting RelA

    PubMed Central

    Choi, Doo Chul; Chae, Yoon-Jee; Kabaria, Savan; Chaudhuri, Amrita Datta; Jain, Mohit Raja; Li, Hong; Mouradian, M. Maral

    2014-01-01

    Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial complex I impairment in PD is modeled in vitro by the susceptibility of dopaminergic neurons to the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). In the present study, we demonstrate that microRNA-7 (miR-7), which is expressed in tyrosine hydroxylase-positive nigral neurons in mice and humans, protects cells from MPP+-induced toxicity in dopaminergic SH-SY5Y cells, differentiated human neural progenitor ReNcell VM cells, and primary mouse neurons. RelA, a component of nuclear factor-κB (NF-κB), was identified to be downregulated by miR-7 using quantitative proteomic analysis. Through a series of validation experiments, it was confirmed that RelA mRNA is a target of miR-7 and is required for cell death following MPP+ exposure. Further, RelA mediates MPP+-induced suppression of NF-κB activity, which is essential for MPP+-induced cell death. Accordingly, the protective effect of miR-7 is exerted through relieving NF-κB suppression by reducing RelA expression. These findings provide a novel mechanism by which NF-κB suppression, rather than activation, underlies the cell death mechanism following MPP+ toxicity, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for this disease. PMID:25232110

  19. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    SciTech Connect

    Naranmandura, Hua; Xu, Shi; Koike, Shota; Pan, Li Qiang; Chen, Bin; Wang, Yan Wei; Rehman, Kanwal; Wu, Bin; Chen, Zhe; Suzuki, Noriyuki

    2012-05-01

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

  20. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.

    PubMed

    Maya-Monteiro, Clarissa M; Almeida, Patricia E; D'Avila, Heloisa; Martins, Aline S; Rezende, Ana Paula; Castro-Faria-Neto, Hugo; Bozza, Patricia T

    2008-01-25

    Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases. PMID:18039669

  1. Evolutionary rewiring of gene regulatory network linkages at divergence of the echinoid subclasses

    PubMed Central

    Erkenbrack, Eric M.; Davidson, Eric H.

    2015-01-01

    Evolution of animal body plans occurs with changes in the encoded genomic programs that direct development, by alterations in the structure of encoded developmental gene-regulatory networks (GRNs). However, study of this most fundamental of evolutionary processes requires experimentally tractable, phylogenetically divergent organisms that differ morphologically while belonging to the same monophyletic clade, plus knowledge of the relevant GRNs operating in at least one of the species. These conditions are met in the divergent embryogenesis of the two extant, morphologically distinct, echinoid (sea urchin) subclasses, Euechinoidea and Cidaroidea, which diverged from a common late Paleozoic ancestor. Here we focus on striking differences in the mode of embryonic skeletogenesis in a euechinoid, the well-known model Strongylocentrotus purpuratus (Sp), vs. the cidaroid Eucidaris tribuloides (Et). At the level of descriptive embryology, skeletogenesis in Sp and Et has long been known to occur by distinct means. The complete GRN controlling this process is known for Sp. We carried out targeted functional analyses on Et skeletogenesis to identify the presence, or demonstrate the absence, of specific regulatory linkages and subcircuits key to the operation of the Sp skeletogenic GRN. Remarkably, most of the canonical design features of the Sp skeletogenic GRN that we examined are either missing or operate differently in Et. This work directly implies a dramatic reorganization of genomic regulatory circuitry concomitant with the divergence of the euechinoids, which began before the end-Permian extinction. PMID:26170318

  2. Zooming-in on cancer metabolic rewiring with tissue specific constraint-based models.

    PubMed

    Di Filippo, Marzia; Colombo, Riccardo; Damiani, Chiara; Pescini, Dario; Gaglio, Daniela; Vanoni, Marco; Alberghina, Lilia; Mauri, Giancarlo

    2016-06-01

    The metabolic rearrangements occurring in cancer cells can be effectively investigated with a Systems Biology approach supported by metabolic network modeling. We here present tissue-specific constraint-based core models for three different types of tumors (liver, breast and lung) that serve this purpose. The core models were extracted and manually curated from the corresponding genome-scale metabolic models in the Human Metabolic Atlas database with a focus on the pathways that are known to play a key role in cancer growth and proliferation. Along similar lines, we also reconstructed a core model from the original general human metabolic network to be used as a reference model. A comparative Flux Balance Analysis between the reference and the cancer models highlighted both a clear distinction between the two conditions and a heterogeneity within the three different cancer types in terms of metabolic flux distribution. These results emphasize the need for modeling approaches able to keep up with this tumoral heterogeneity in order to identify more suitable drug targets and develop effective treatments. According to this perspective, we identified key points able to reverse the tumoral phenotype toward the reference one or vice-versa. PMID:27085310

  3. Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma

    PubMed Central

    2013-01-01

    Background We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL. Methods Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry. Results We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed. Conclusions This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis. PMID:23422267

  4. Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis*

    PubMed Central

    Dhruv, Harshil; Loftus, Joseph C.; Narang, Pooja; Petit, Joachim L.; Fameree, Maureen; Burton, Julien; Tchegho, Giresse; Chow, Donald; Yin, Holly; Al-Abed, Yousef; Berens, Michael E.; Tran, Nhan L.; Meurice, Nathalie

    2013-01-01

    Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr176, but not Trp231, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr176 as the anchoring residue. Importantly, mutation of TWEAK at Tyr176 did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers. PMID:24056367

  5. Mir-302c mediates influenza A virus-induced IFNβ expression by targeting NF-κB inducing kinase.

    PubMed

    Gui, Shulin; Chen, Xueyuan; Zhang, Mo; Zhao, Fanpeng; Wan, Yushun; Wang, Li; Xu, Gang; Zhou, Li; Yue, Xin; Zhu, Ying; Liu, Shi

    2015-12-21

    Little is known about the role of microRNA during influenza A virus (IAV) infection. We observed that NIK 3'UTR luciferase activity was elevated during IAV infection. Further studies demonstrated that miR-302c reduced NIK expression, resulting in the reduction of IFNβ mRNA expression. We found that miR-302c prevented the translocation of NF-κB from the cytosol to the nucleus. Furthermore, IAV infection downregulated miR-302c expression, leading to the activation of IFNβ expression and the inhibition of viral replication. Compared to miR-302c, miR-520e cannot promote viral replication and production, although the two microRNAs target the same site of the NIK 3'UTR. Collectively, our work defines a novel signaling pathway implicated in the control of IFNβ mRNA expression during IAV infection. PMID:26602079

  6. Measurements of neutron emission induced by muons stopped in metal deuteride targets

    NASA Astrophysics Data System (ADS)

    Chen, M.; Steadman, S. G.; Gaudreau, M. P. J.; Luckhardt, S. C.; Parker, R. R.; Albagli, D.; Cammarata, V.; Schloh, M.; Wrighton, M. S.; Kwok, K.; Thieme, C.; Lowenstein, D. I.; Debbe, R.; Reilly, J. J.

    1990-06-01

    An 80-MeV/c negative muon beam from the Alternating Gradient Synchrotron at Brookhaven National Laboratory was used to investigate the stopping of muons inside Pd, Ti, and Y targets saturated with deuterium. Neutron emission from the targets was measured with an array of3He detectors, and in some runs, the temperature of the target was monitored as a function of time, with and without a flux of muons on the target. The neutron rates were also measured for Pd cathodes in an active electrochemical cell similar in design to those used in so-called “cold fusion” experiments, and the electrolyte solution was analyzed for excess tritium. No evidence was found for muon-catalyzed fusion at rates consistent with those claimed in “cold fusion” experiments. Neutron production from catalyzed fusion due to the presence of deuterium in palladium deuteride, PdD0.7, exposed to muons was determined to be 0.0±0.03 (stat.) ±0.25 (syst.) neutrons per stopped muon.

  7. IRF1 marks activated genes in SLE and can induce target gene expression

    PubMed Central

    Zhang, Zhe; Shi, Lihua; Song, Li; Ephrem, Elshaddai; Petri, Michelle; Sullivan, Kathleen E.

    2014-01-01

    Objective IRF1 both mediates responses to type I interferons and the induction of interferons. It has been implicated in murine lupus models as a critical mediator of inflammation. A previous study of chromatin modifications in SLE patient monocytes implicated IRF1 as associated with increased histone acetylation in SLE patients. This study directly investigated IRF1 binding sites on chromatin using ChIP-seq. Methods Nine female SLE patients and seven female controls were examined. Monocytes were purified from peripheral blood and subjected to library preparation using a validated antibody to IRF1. The effect of IRF1 on target gene expression was confirmed using an overexpression system in cell lines and co-immunoprecipitation was used to define protein interactions. Results IRF1 binding around transcribed regions was increased in SLE patient monocytes but histone modifications at potential IRF1 binding sites without detectable IRF1 binding were also increased. IRF1 overexpression was sufficient to drive transcription of target genes. IRF1 overexpression was also able to alter histone modifications at a focus set of target genes and the use of an IRF1 inhibitor decreased both expression and histone modifications at target genes. IRF1 was found to interact with a select set of histone modifying enzymes and other transcription factors. Conclusions IRF1 is an important signaling protein in the interferon pathway. IRF1 not only activates gene expression as a transcription factor but may perpetuate disease by leading to a dysregulated epigenome. PMID:25418955

  8. Targeting Thioredoxin Reductase by Parthenolide Contributes to Inducing Apoptosis of HeLa Cells.

    PubMed

    Duan, Dongzhu; Zhang, Junmin; Yao, Juan; Liu, Yaping; Fang, Jianguo

    2016-05-01

    Parthenolide (PTL), a major active sesquiterpene lactone from the herbal plant Tanacetum parthenium, has been applied in traditional Chinese medicine for centuries. Although PTL demonstrates potent anticancer efficacy in numerous types of malignant cells, the cellular targets of PTL have not been well defined. We reported here that PTL interacts with both cytosolic thioredoxin reductase (TrxR1) and mitochondrial thioredoxin reductase (TrxR2), two ubiquitous selenocysteine-containing antioxidant enzymes, to elicit reactive oxygen species-mediated apoptosis in HeLa cells. PTL selectively targets the selenocysteine residue in TrxR1 to inhibit the enzyme function, and further shifts the enzyme to an NADPH oxidase to generate superoxide anions, leading to reactive oxygen species accumulation and oxidized thioredoxin. Under the conditions of inhibition of TrxRs in cells, PTL does not cause significant alteration of cellular thiol homeostasis, supporting selective target of TrxRs by PTL. Importantly, overexpression of functional TrxR1 or Trx1 confers protection, whereas knockdown of the enzymes sensitizes cells to PTL treatment. Targeting TrxRs by PTL thus discloses an unprecedented mechanism underlying the biological activity of PTL, and provides deep insights to understand the action of PTL in treatment of cancer. PMID:27002142

  9. Measurements of neutron emission induced by muons stopped in metal deuteride targets

    SciTech Connect

    Chen, M.; Steadman, S.G.; Gaudreau, M.P.J.; Luckhardt, S.C.; Parker, R.R.; Albagli, D.; Cammarata, V.; Schloh, M.; Wrighton, M.S.; Kwok, K.; Thieme, C.; Lowenstein, D.I.; Debbe, R.; Reilly, J.J.; Brookhaven National Lab., Upton, NY )

    1989-01-01

    An 80 MeV/c negative muon beam from the Alternating Gradient Synchrotron at Brookhaven National Laboratory was used to investigate the stopping of muons inside Pd, Ti and Y targets saturated with deuterium. Neutron emission from the targets was measured with an array of {sup 3}He detectors, and in some runs, the temperature of the target was monitored as a function of time, with and without a flux of muons on the target. The neutron rates were also measured for Pd cathodes in an active electrochemical cell similar in design to those used in so-called cold-fusion'' experiments, and the electrolyte solution was analyzed for excess tritium at rates consistent with these claimed in cold fusion'' experiments. Neutron production catalyzed fusion due to the presence of deuterium in palladium deuteride, PdD{sub 0.7}, exposed to muons was determined in palladium 0.0 {plus minus} 0.03 (stat.) {plus minus} 0.25 (syst.) neutrons per stored muon. 15 refs., 5 figs.

  10. Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalisation without inducing empty basement membrane sleeves in xenograft tumours

    PubMed Central

    Coutelle, O; Schiffmann, L M; Liwschitz, M; Brunold, M; Goede, V; Hallek, M; Kashkar, H; Hacker, U T

    2015-01-01

    Background: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS. Methods: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg−1) or high (5 mg kg−1) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation. Results: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. Conclusions: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS. PMID:25562438

  11. Unveiling the identity of distant targets through advanced Raman-laser-induced breakdown spectroscopy data fusion strategies.

    PubMed

    Moros, Javier; Laserna, J Javier

    2015-03-01

    Data fusion is the process of combining data gathered from two or more sensors to produce a more specific, comprehensive and unified dataset of the inspected target. On this basis, much has been said about the possible benefits resulting from the use of molecular and atomic information for the detection of explosives. The orthogonal nature of the spectral and compositional information provided by Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS) makes them suitable candidates for an optimal combination of their data, thus achieving inferences that are not feasible using a single sensor. The present manuscript evaluates several architectures for the combination of spectral outputs from these two sensors in order to compare the benefits and drawbacks of data fusion for improving the overall identification performance. From the simple assembling (concatenation or addition) of Raman and LIBS spectra to signals' processing on the basis of linear algebra (either the outer product or the outer sum), different identification patterns of several compounds (explosives, potential confusants and supports) have been built. The efficiency on target differentiation by using each of the architectures has been evaluated by comparing the identification yield obtained for all the inspected targets from correlation and similarity measurements. Additionally, a specific code integrated by several of these patterns to identify each compound has also been evaluated. This approach permits to obtain a better knowledge about the identity of an interrogated target, mainly in those decisive cases in which LIBS or Raman cannot be effective separately to reach a decision. PMID:25618716

  12. Induced radioactivity in CU targets produced by high-energy heavy ions and the corresponding estimated photon dose rates.

    PubMed

    Yashima, H; Uwamino, Y; Sugita, H; Ito, S; Nakamura, T; Fukumura, A

    2004-01-01

    Irradiation experiments were performed at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility, National Institute of Radiological Sciences. The radioactive spallation products in a thick Cu target were obtained for Ar(230, 400 MeV per nucleon), Si(800 MeV per nucleon), Ne(100, 230, 400 MeV per nucleon), C(100, 230, 400 MeV per nucleon), He(100, 230 MeV per nucleon), p(100, 230 MeV) ions. The gamma-ray spectra from irradiated Cu samples inserted into the composite Cu target were measured with a high-purity germanium (HPGe) detector. From the gamma-ray spectra, we obtained the spatial distribution of radioactive yields of spallation products of 40 nuclides in the Cu sample in the Cu target. From the spatial distribution of radioactive yields, we estimated the residual activity and photon dose induced in the Cu target. The residual activity and photon dose become larger with the increase in projectile energy per nucleon and the range of the projectile beam for the same projectile energy per nucleon. PMID:15280565

  13. Tangeretin Alleviates Cisplatin-Induced Acute Hepatic Injury in Rats: Targeting MAPKs and Apoptosis

    PubMed Central

    Omar, Hany A.; Mohamed, Wafaa R.; Arab, Hany H.; Arafa, El-Shaimaa A.

    2016-01-01

    Despite its broad applications, cisplatin affords considerable nephro- and hepatotoxicity through triggering inflammatory and oxidative stress cascades. The aim of the current investigation was to study the possible protective effects of tangeretin on cisplatin-induced hepatotoxicity. The impact of tangeretin on cisplatin-evoked hepatic dysfunction and histopathologic changes along with oxidative stress, inflammatory and apoptotic biomarkers were investigated compared to silymarin. Tangeretin pre-treatment significantly improved liver function tests (ALT and AST), inhibited cisplatin-induced lipid profile aberrations (total cholesterol and triglycerides) and diminished histopathologic structural damage in liver tissues. Tangeretin also attenuated cisplatin-induced hepatic inflammatory events as indicated by suppression of tumor necrosis factor-α (TNF-α) and enhancement of interleukin-10 (IL-10). Meanwhile, it lowered malondialdehyde (MDA), nitric oxide (NO) and nuclear factor erythroid 2-related factor 2 (NRF-2) levels with restoration of glutathione (GSH), and glutathione peroxidase (GPx). Regarding mitogen-activated protein kinase (MAPK) pathway, tangeretin attenuated cisplatin-induced increase in phospho-p38, phospho-c-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinase (p-ERK1/2) in liver tissues. In addition, tangeretin downregulated Bax expression with augmentation of Bcl-2 promoting liver cell survival. Our results highlight the protective effects of tangeretin against cisplatin-induced acute hepatic injury via the concerted modulation of inflammation, oxidative stress, MAPKs and apoptotic pathways. PMID:27031695

  14. Tangeretin Alleviates Cisplatin-Induced Acute Hepatic Injury in Rats: Targeting MAPKs and Apoptosis.

    PubMed

    Omar, Hany A; Mohamed, Wafaa R; Arab, Hany H; Arafa, El-Shaimaa A

    2016-01-01

    Despite its broad applications, cisplatin affords considerable nephro- and hepatotoxicity through triggering inflammatory and oxidative stress cascades. The aim of the current investigation was to study the possible protective effects of tangeretin on cisplatin-induced hepatotoxicity. The impact of tangeretin on cisplatin-evoked hepatic dysfunction and histopathologic changes along with oxidative stress, inflammatory and apoptotic biomarkers were investigated compared to silymarin. Tangeretin pre-treatment significantly improved liver function tests (ALT and AST), inhibited cisplatin-induced lipid profile aberrations (total cholesterol and triglycerides) and diminished histopathologic structural damage in liver tissues. Tangeretin also attenuated cisplatin-induced hepatic inflammatory events as indicated by suppression of tumor necrosis factor-α (TNF-α) and enhancement of interleukin-10 (IL-10). Meanwhile, it lowered malondialdehyde (MDA), nitric oxide (NO) and nuclear factor erythroid 2-related factor 2 (NRF-2) levels with restoration of glutathione (GSH), and glutathione peroxidase (GPx). Regarding mitogen-activated protein kinase (MAPK) pathway, tangeretin attenuated cisplatin-induced increase in phospho-p38, phospho-c-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinase (p-ERK1/2) in liver tissues. In addition, tangeretin downregulated Bax expression with augmentation of Bcl-2 promoting liver cell survival. Our results highlight the protective effects of tangeretin against cisplatin-induced acute hepatic injury via the concerted modulation of inflammation, oxidative stress, MAPKs and apoptotic pathways. PMID:27031695

  15. Hypoxia inducible factor prolyl hydroxylases as targets for neuroprotection by “antioxidant” metal chelators: from ferroptosis to stroke

    PubMed Central

    Speer, Rachel E.; Karuppagounder, Saravanan S.; Basso, Manuela; Sleiman, Sama; Kumar, Amit; Brand, David; Smirnova, Natalya; Gazaryan, Irina; Khim, Soah J.; Ratan, Rajiv R.

    2015-01-01

    Neurologic conditions including stroke, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are leading causes of death and long-term disability in the United States, and efforts to develop novel therapeutics for these conditions have historically had poor success in translating from bench to bedside. Hypoxia Inducible Factor-1alpha (HIF-1α) mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway are neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adpatation in detail, and provide perspective on which targets within this pathway appear to be ripest for preventing and repairing neurodegeneration. Further, we highlight the role of HIF prolyl hydroxylases as emerging targets for the salutary effects of metal chelators on ferroptosis in vitro as well in animal models of neurological diseases. PMID:23376032

  16. Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance

    PubMed Central

    Gabriel, Sarah Sharon; Bon, Nina; Chen, Jin; Wekerle, Thomas; Bushell, Andrew; Fehr, Thomas; Cippà, Pietro Ernesto

    2016-01-01

    Distinctive molecular characteristics of functionally diverse lymphocyte populations may represent novel pharmacological targets for immunotherapy. The intrinsic apoptosis pathway is differently regulated among conventional and regulatory T cells (Tregs). Targeted pharmacological modulation of this pathway with a small molecule Bcl-2/Bcl-xL inhibitor (ABT-737) caused a selective depletion of effector T cells and a relative enrichment of Tregs in vivo. Treatment with ABT-737 resulted in a tolerogenic milieu, which was exploited to alleviate graft-versus-host disease, to prevent allograft rejection in a stringent fully MHC-mismatched skin transplantation model and to induce immunological tolerance in combination with bone marrow transplantation. This concept has the potential to find various applications for immunotherapy, since it allows pharmacologic exploitation of the immunomodulatory properties of Tregs without the need for cell manipulation ex vivo. PMID:26973650

  17. Recent advances and new perspectives in targeting CFTR for therapy of cystic fibrosis and enterotoxin-induced secretory diarrheas

    PubMed Central

    Zhang, Weiqiang; Fujii, Naoaki; Naren, Anjaparavanda P

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical surfaces of epithelial cells lining airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with an altered channel function of CFTR. Cystic fibrosis (CF) is caused by the loss or dysfunction of CFTR-channel activity resulting from the mutations on the gene; whereas enterotoxin-induced secretory diarrheas are caused by the hyperactivation of CFTR channel function. CFTR is a validated target for drug development to treat these diseases. Significant progress has been made in developing CFTR modulator therapy by means of high-throughput screening followed by hit-to-lead optimization. Several oral administrated investigational drugs are currently being evaluated in clinical trials for CF. Also importantly, new ideas and methodologies are emerging. Targeting CFTR-containing macromolecular complexes is one such novel approach. PMID:22393940

  18. Comparison between experiments and molecular dynamic simulations of spallation induced by ultra-short laser shock on micrometric Tantalum targets

    NASA Astrophysics Data System (ADS)

    Cuq-Lelandais, Jean-Paul; Boustie, Michel; Soulard, Laurent; Berthe, Laurent; Sollier, Arnaud; Bontaz-Carion, Joelle; Combis, Patrick; de Resseguier, Thibaut; Lescoute, Emilien

    2009-06-01

    Shock wave propagation and the spallation within materials induced by laser shock have been investigated for roughly two decades. With the latest laser technologies evolution, one can access to shorter regimes in durations, going below the picosecond range. Shots performed with the LULI 100TW facility evidence the possibility to obtain spallation in a few microns thick metallic target. Such conditions provide an experimental data layout directly comparable with molecular dynamic simulations accessible to these scales. Molecular dynamic simulations on a single crystal of Tantalum have been performed with the CEA TERA 10 computer. First, the Hugoniot calculated by the equilibrium molecular dynamics has been compared with experimental data to check the potential (EAM) relevance to reproduce the shock wave propagation. Then, a large scale simulation on a micrometric target has been performed. We have observed the microscopic ductile damage process, the pore apparition and their time and space evolution. The results are compared with experimental results and classical one- dimensional hydrodynamic simulations.

  19. Spatial characterization of the laser-induced plasma plumes generated by IR CO2 pulsed laser on carbon targets

    NASA Astrophysics Data System (ADS)

    Camacho, J. J.; Santos, M.; Díaz, L.; Poyato, J. M. L.

    2009-02-01

    Spatially resolved optical emission analysis was carried out for the plasma plume, produced by high-power tunable IR CO2 pulsed laser ablation of graphite, at λ=9.621 μm and with laser fluence of 342 J cm-2. Wavelength-dispersed spectra of the plume, at medium vacuum conditions ( P air=4 Pa) and concentrated close to the target, reveal C, C+, C2+, C3+, C4+, N, H, O, and molecular emissions between different electronic states of C2, CN, OH, CH, and NH. The characteristics of the spectral emission intensities from different species have been investigated as functions of the distance (up to 20 cm) from the sample surface. Vibrational temperatures in the laser-induced plasma have been estimated at various distances from the target surface.

  20. MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

    SciTech Connect

    Cha, Min-Ji; Jang, Jin-Kyung; Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon; Lee, Chang Yeon; Park, Jun-Hee; Lee, Jiyun; Seo, Hyang-Hee; Choi, Eunhyun; Jeon, Woo-min; Hwang, Hye Jin; Shin, Hyun-Taek; and others

    2013-06-14

    Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

  1. Therapeutic Antibody-Induced Vascular Toxicity Due to Off-Target Activation of Nitric Oxide in Cynomolgus Monkeys.

    PubMed

    Pai, Rama; Ma, Ning; Connor, Anu V; Danilenko, Dimitry M; Tarrant, Jacqueline M; Salvail, Dany; Wong, Lisa; Hartley, Dylan P; Misner, Dinah; Stefanich, Eric; Wu, Yan; Chen, Yongmei; Wang, Hong; Dambach, Donna M

    2016-06-01

    PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic

  2. Aberrant excitatory rewiring of layer V pyramidal neurons early after neocortical trauma.

    PubMed

    Takahashi, D Koji; Gu, Feng; Parada, Isabel; Vyas, Shri; Prince, David A

    2016-07-01

    Lesioned neuronal circuits form new functional connections after a traumatic brain injury (TBI). In humans and animal models, aberrant excitatory connections that form after TBI may contribute to the pathogenesis of post-traumatic epilepsy. Partial neocortical isolation ("undercut" or "UC") leads to altered neuronal circuitry and network hyperexcitability recorded in vivo and in brain slices from chronically lesioned neocortex. Recent data suggest a critical period for maladaptive excitatory circuit formation within the first 3days post UC injury (Graber and Prince 1999, 2004; Li et al. 2011, 2012b). The present study focuses on alterations in excitatory connectivity within this critical period. Immunoreactivity (IR) for growth-associated protein (GAP)-43 was increased in the UC cortex 3days after injury. Some GAP-43-expressing excitatory terminals targeted the somata of layer V pyramidal (Pyr) neurons, a domain usually innervated predominantly by inhibitory terminals. Immunocytochemical analysis of pre- and postsynaptic markers showed that putative excitatory synapses were present on somata of these neurons in UC neocortex. Excitatory postsynaptic currents from UC layer V Pyr cells displayed properties consistent with perisomatic inputs and also reflected an increase in the number of synaptic contacts. Laser scanning photostimulation (LSPS) experiments demonstrated reorganized excitatory connectivity after injury within the UC. Concurrent with these changes, spontaneous epileptiform bursts developed in UC slices. Results suggest that aberrant reorganization of excitatory connectivity contributes to early neocortical hyperexcitability in this model. The findings are relevant for understanding the pathophysiology of neocortical post-traumatic epileptogenesis and are important in terms of the timing of potential prophylactic treatments. PMID:26956396

  3. Target geometry and rigidity determines laser-induced cavitation bubble transport and nanoparticle productivity - a high-speed videography study.

    PubMed

    Kohsakowski, Sebastian; Gökce, Bilal; Tanabe, Rie; Wagener, Philipp; Plech, Anton; Ito, Yoshiro; Barcikowski, Stephan

    2016-06-28

    Laser-induced cavitation has mostly been studied in bulk liquid or at a two-dimensional wall, although target shapes for the particle synthesis may strongly affect bubble dynamics and interfere with particle productivity. We investigated the dynamics of the cavitation bubble induced by pulsed-laser ablation in liquid for different target geometries with high-speed laser microsecond videography and focus on the collapse behaviour. This method enables us observations in a high time resolution (intervals of 1 μs) and single-pulse experiments. Further, we analyzed the nanoparticle productivity, the sizes of the synthesized nanoparticles and the evolution of the bubble volume for each different target shape and geometry. For the ablation of metal (Ag, Cu, Ni) wire tips a springboard-like behaviour after the first collapse is observed which can be correlated with vertical projectile motion. Its turbulent friction in the liquid causes a very efficient transport and movement of the bubble and ablated material into the bulk liquid and prevents particle redeposition. This effect is influenced by the degree of freedom of the wire as well as the material properties and dimensions, especially the Young's modulus. The most efficient and largest bubble movement away from the wire was observed for a thin (500 μm) silver wire with velocities up to 19.8 m s(-1) and for materials with a small Young's modulus and flexural rigidity. We suggest that these observations may contribute to upscaling strategies and increase of particle yield towards large synthesis of colloids based on targets that may continuously be fed. PMID:27273693

  4. Targeting of Fn14 Prevents Cancer-Induced Cachexia and Prolongs Survival.

    PubMed

    Johnston, Amelia J; Murphy, Kate T; Jenkinson, Laura; Laine, David; Emmrich, Kerstin; Faou, Pierre; Weston, Ross; Jayatilleke, Krishnath M; Schloegel, Jessie; Talbo, Gert; Casey, Joanne L; Levina, Vita; Wong, W Wei-Lynn; Dillon, Helen; Sahay, Tushar; Hoogenraad, Joan; Anderton, Holly; Hall, Cathrine; Schneider, Pascal; Tanzer, Maria; Foley, Michael; Scott, Andrew M; Gregorevic, Paul; Liu, Spring Yingchun; Burkly, Linda C; Lynch, Gordon S; Silke, John; Hoogenraad, Nicholas J

    2015-09-10

    The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients. PMID:26359988

  5. Calculations of Neutron- and Proton-Induced Reactions up to 200 MeV for Target 238U

    SciTech Connect

    Yu Hongwei; Zhao Zhixiang; Cai Chonghai

    2005-05-24

    The calculations of neutron- and proton-induced reaction up to 200 MeV for target 238U are performed; the calculated results are generally in good agreement with experimental data, and the physics is rational. The theoretical framework consists of the spherical optical model, intranuclear cascade mechanism for nucleon emission based on empirical formula, preequilibrium emission theory based on exciton model, evaporation model, and Hauser-Feshbach statistical theory with a width fluctuation correction. The fission widths are calculated using the Bohr-Wheeler formula.

  6. Reaction dynamics induced by the radioactive ion beam 7Be on medium-mass and heavy targets

    NASA Astrophysics Data System (ADS)

    Mazzocco, M.; Boiano, A.; Boiano, C.; La Commara, M.; Manea, C.; Parascandolo, C.; Pierroutsakou, D.; Stefanini, C.; Strano, E.; Torresi, D.; Acosta, L.; Di Meo, P.; Fernandez-Garcia, J. P.; Glodariu, T.; Grebosz, J.; Guglielmetti, A.; Keeley, N.; Lay, J. A.; Marquinez-Duran, G.; Martel, I.; Mazzocchi, C.; Molini, P.; Nicoletto, M.; Pakou, A.; Parkar, V. V.; Rusek, K.; Sánchez-Benítez, A. M.; Sandoli, M.; Sava, T.; Sgouros, O.; Signorini, C.; Silvestri, R.; Soramel, F.; Soukeras, V.; Stiliaris, E.; Stroe, L.; Toniolo, N.; Zerva, K.

    2015-10-01

    We studied the reaction dynamics induced at Coulomb barrier energies by the weakly-bound Radioactive Ion Beam 7Be (Sα = 1.586 MeV) on medium-mass (58Ni) and heavy (208Pb) targets. The experiments were performed at INFN-LNL (Italy), where a 2-3×105 pps 7Be secondary beam was produced with the RIB in-flight facility EXOTIC. Charged reaction products were detected by means of high-granularity silicon detectors in rather wide angular ranges. The contribution presents an up-to-date status of the data analysis and theoretical interpretation for both systems.

  7. Conophylline protects cells in cellular models of neurodegenerative diseases by inducing mammalian target of rapamycin (mTOR)-independent autophagy.

    PubMed

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-03-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  8. Conophylline Protects Cells in Cellular Models of Neurodegenerative Diseases by Inducing Mammalian Target of Rapamycin (mTOR)-independent Autophagy*

    PubMed Central

    Sasazawa, Yukiko; Sato, Natsumi; Umezawa, Kazuo; Simizu, Siro

    2015-01-01

    Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD. PMID:25596530

  9. Location and detection of explosive-contaminated human fingerprints on distant targets using standoff laser-induced breakdown spectroscopy

    NASA Astrophysics Data System (ADS)

    Lucena, P.; Gaona, I.; Moros, J.; Laserna, J. J.

    2013-07-01

    Detection of explosive-contaminated human fingerprints constitutes an analytical challenge of high significance in security issues and in forensic sciences. The use of a laser-induced breakdown spectroscopy (LIBS) sensor working at 31 m distance to the target, fitted with 2D scanning capabilities and designed for capturing spectral information from laser-induced plasmas of fingerprints is presented. Distribution chemical maps based on Na and CN emissions are used to locate and detect chloratite, DNT, TNT, RDX and PETN residues that have been deposited on the surface of aluminum and glass substrates. An effectiveness of 100% on fingerprints detection, regardless the substrate scanned, is reached. Environmental factors that affect the prevalence of the fingerprint LIBS response are discussed.

  10. Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

    PubMed

    Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F

    2016-01-01

    Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization. PMID:26725118

  11. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    SciTech Connect

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R. . E-mail: nerurkar@pbrc.hawaii.edu

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

  12. Running rewires the neuronal network of adult-born dentate granule cells.

    PubMed

    Vivar, Carmen; Peterson, Benjamin D; van Praag, Henriette

    2016-05-01

    Exercise improves cognition in humans and animals. Running increases neurogenesis in the dentate gyrus of the hippocampus, a brain area important for learning and memory. It is unclear how running modifies the circuitry of new dentate gyrus neurons to support their role in memory function. Here we combine retroviral labeling with rabies virus mediated trans-synaptic retrograde tracing to define and quantify new neuron afferent inputs in young adult male C57Bl/6 mice, housed with or without a running wheel for one month. Exercise resulted in a shift in new neuron networks that may promote sparse encoding and pattern separation. Neurogenesis increased in the dorsal, but not the ventral, dentate gyrus by three-fold, whereas afferent traced cell labeling doubled in number. Regional analysis indicated that running differentially affected specific inputs. Within the hippocampus the ratio of innervation from inhibitory interneurons and glutamatergic mossy cells to new neurons was reduced. Distal traced cells were located in sub-cortical and cortical regions, including perirhinal, entorhinal and sensory cortices. Innervation from entorhinal cortex (EC) was augmented, in proportion to the running-induced enhancement of adult neurogenesis. Within EC afferent input and short-term synaptic plasticity from lateral entorhinal cortex, considered to convey contextual information to the hippocampus was increased. Furthermore, running upregulated innervation from regions important for spatial memory and theta rhythm generation, including caudo-medial entorhinal cortex and subcortical medial septum, supra- and medial mammillary nuclei. Altogether, running may facilitate contextual, spatial and temporal information encoding by increasing adult hippocampal neurogenesis and by reorganization of new neuron circuitry. PMID:26589333

  13. Recent progress in stimuli-induced polydiacetylenes for sensing temperature, chemical and biological targets.

    PubMed

    Lee, Songyi; Kim, Ji-Yeong; Chen, Xiaoqiang; Yoon, Juyoung

    2016-07-28

    Polydiacetylenes (PDAs) have received increasing attention as smart materials owing to their unique properties. Upon addition of various stimuli, blue PDAs can undergo a colorimetric transition from blue to red along with a change from non-fluorescent to fluorescent. The optical changes can be readily detected by the naked eye and by using absorption and fluorescence spectrometers. These properties make PDAs excellent materials for use in platforms for sensing chemical or biological targets. In recent years, a number of biosensors and chemosensors based on the optical responses of polydiacetylenes have been reported. In this review, recent advances made in this area were discussed following a format based on different cognizing targets, including temperature, metal ions, anions, surfactants, amines, water, gas, sugars, hydrocarbons, neomycin, heparin, virus, enzymes, bacteria, and cancers. Emphasis is given to the methods used to prepare PDA sensing systems as well as their sensing performance. PMID:27314281

  14. Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms

    PubMed Central

    Wen, Qiang Jeremy; Yang, Qiong; Goldenson, Benjamin; Malinge, Sébastien; Lasho, Terra; Schneider, Rebekka K.; Breyfogle, Lawrence J.; Schultz, Rachael; Gilles, Laure; Koppikar, Priya; Abdel-Wahab, Omar; Pardanani, Animesh; Stein, Brady; Gurbuxani, Sandeep; Mullally, Ann; Levine, Ross; Tefferi, Ayalew; Crispino, John D.

    2015-01-01

    Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF. PMID:26569382

  15. Therapeutic Targeting of CPT-11 Induced Diarrhea: A Case for Prophylaxis

    PubMed Central

    Swami, Umang; Goel, Sanjay; Mani, Sridhar

    2014-01-01

    CPT-11 (irinotecan), a DNA topoisomerase I inhibitor is one of the main treatments for colorectal cancer. The main dose limiting toxicities are neutropenia and late onset diarrhea. Though neutropenia is manageable, CPT-11 induced diarrhea is frequently severe, resulting in hospitalizations, dose reductions or omissions leading to ineffective treatment administration. Many potential agents have been tested in preclinical and clinical studies to prevent or ameliorate CPT-11 induced late onset diarrhea. It is predicted that prophylaxis of CPT-11 induced diarrhea will reduce sub-therapeutic dosing as well as hospitalizations and will eventually lead to dose escalations resulting in better response rates. This article reviews various experimental agents and strategies employed to prevent this debilitating toxicity. Covered topics include schedule/dose modification, intestinal alkalization, structural/chemical modification, genetic testing, anti-diarrheal therapies, transporter (ABCB1, ABCC2, BCRP2) inhibitors, enzyme (β-glucuronidase, UGT1A1, CYP3A4, carboxylesterase, COX-2) inducers and inhibitors, probiotics, antibiotics, adsorbing agents, cytokine and growth factor activators and inhibitors and other miscellaneous agents. PMID:23597015

  16. Rescuing Stimuli from Invisibility: Inducing a Momentary Release from Visual Masking with Pre-Target Entrainment

    ERIC Educational Resources Information Center

    Mathewson, Kyle E.; Fabiani, Monica; Gratton, Gabriele; Beck, Diane M.; Lleras, Alejandro

    2010-01-01

    At near-threshold levels of stimulation, identical stimulus parameters can result in very different phenomenal experiences. Can we manipulate which stimuli reach consciousness? Here we show that consciousness of otherwise masked stimuli can be experimentally induced by sensory entrainment. We preceded a backward-masked stimulus with a series of…

  17. Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...

  18. Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

    SciTech Connect

    Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan; Reddy, Kanamata; Kurinov, Igor; Carper, Andria; Langfald, Alyssa; Oi, Naomi; Kim, Myoung Ok; Zhu, Feng; Sosa, Carlos P.; Zhou, Keyuan; Bode, Ann M.; Dong, Zigang

    2012-06-27

    Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

  19. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1{alpha} survival pathways

    SciTech Connect

    Oommen, Deepu; Prise, Kevin M.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer KNK437, a benzylidene lactam compound, is a novel radiosensitizer. Black-Right-Pointing-Pointer KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1{alpha} under hypoxia. Black-Right-Pointing-Pointer KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1{alpha} (HIF-1{alpha}). HIF-1{alpha} is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1{alpha}. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1{alpha} in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1{alpha} levels in KNK437-treated cells. This suggested that the absence of HIF-1{alpha} in hypoxic cells was not due to the enhanced protein degradation. HIF-1{alpha} is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1{alpha} mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1{alpha} levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  20. Targeted inhibition of p38MAPK-enhanced autophagy in SW620 cells resistant to photodynamic therapy-induced apoptosis.

    PubMed

    Xue, Qin; Wang, Pan; Wang, Xiaobing; Zhang, Kun; Liu, Quanhong

    2015-09-01

    Photodynamic therapy (PDT) is a promising and noninvasive treatment that can induce apoptosis, autophagy, or both depending on the cell phenotype. In this work, chlorin e6 (Ce6) was used to photosensitize human colorectal cancer SW620 cells. In cells, apparent autophagy and apoptosis with dependence on intracellular reactive oxygen species (ROS) generation were detected. p38MAPK activation followed by ROS generation might be a core component in Ce6 mediate PDT (Ce6-PDT)-induced autophagy and apoptosis signaling pathway. By using p38MAPK siRNA, the results showed a marked enhancement on cell apoptosis in Ce6-PDT with increased annexin (+) apoptotic cells, nuclear condensation, caspase-3, and PARP cleavage. Besides, impairment of p38MAPK also promoted the autophagic response to photodamage as indicated by conversion of LC3 and monodansyl cadaverine (MDC) labeling patterns. It appears that Ce6-PDT induced ROS production involving activation of p38MAPK, probably to prevent SW620 cells from photodamage. Moreover, autophagy inhibitor 3-methyladenine/bafilomycin A1 greatly aggravated Ce6-PDT-induced apoptosis in SW620 cells with knockdown of p38MAPK. Taken together, this study suggests that autophagy could represent a promising field in cancer treatment and p38MAPK may be a potential therapeutic target to enhance the efficacy on clinical evaluation for the treatment of colorectal cancer. PMID:26254783

  1. Adenovirus-mediated siRNA targeting CXCR2 attenuates titanium particle-induced osteolysis by suppressing osteoclast formation.

    PubMed

    Wang, Chen; Liu, Yang; Wang, Yang; Li, Hao; Zhang, Ran-Xi; He, Mi-Si; Chen, Liang; Wu, Ning-Ning; Liao, Yong; Deng, Zhong-Liang

    2016-01-01

    BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening. PMID:26939934

  2. The ClC-3 chloride channel associated with microtubules is a target of paclitaxel in its induced-apoptosis

    PubMed Central

    Zhang, Haifeng; Li, Huarong; Yang, Lili; Deng, Zhiqin; Luo, Hai; Ye, Dong; Bai, Zhiquan; Zhu, Linyan; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2013-01-01

    Recent evidences show that cationic fluxes play a pivotal role in cell apoptosis. In this study, the roles of Cl− channels in paclitaxel-induced apoptosis were investigated in nasopharyngeal carcinoma CNE-2Z cells. Chloride current and apoptosis were induced by paclitaxel and inhibited by chloride channel blockers. Paclitaxel-activated current possessed similar properties to volume-activated chloride current. After ClC-3 was knocked-down by ClC-3-siRNA, hypotonicity-activated and paclitaxel-induced chloride currents were obviously decreased, indicating that the chloride channel involved in paclitaxel-induced apoptosis may be ClC-3. In early apoptotic cells, ClC-3 was up-regulated significantly; over-expressed ClC-3 was accumulated in cell membrane to form intercrossed filaments, which were co-localized with α-tubulins; changes of ultrastructures and decrease of flexibility in cell membrane were detected by atomic force microscopy. These suggest that ClC-3 is a critical target of paclitaxel and the involvement of ClC-3 in apoptosis may be associated with its accumulation with membrane microtubules and its over activation. PMID:24026363

  3. Adenovirus-Mediated siRNA Targeting CXCR2 Attenuates Titanium Particle-Induced Osteolysis by Suppressing Osteoclast Formation

    PubMed Central

    Wang, Chen; Liu, Yang; Wang, Yang; Li, Hao; Zhang, Ran-Xi; He, Mi-Si; Chen, Liang; Wu, Ning-Ning; Liao, Yong; Deng, Zhong-Liang

    2016-01-01

    Background Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. Material/Methods Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. Results Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. Conclusions CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening. PMID:26939934

  4. NGFI-B targets mitochondria and induces cardiomyocyte apoptosis in restraint-stressed rats by mediating energy metabolism disorder

    PubMed Central

    Wang, XinXing; Liu, XiaoHua; Kong, RuiRui; Zhan, Rui; Wang, XiaoMing; Leng, Xue; Gong, JingBo; Duan, Meng; Wang, LiQun; Wu, Lei

    2009-01-01

    NGFI-B/Nur77/TR3, originally identified as an immediate-early gene rapidly induced by serum and growth factors, is a member of the steroid hormone nuclear receptor superfamily with no identified endogenous ligand. NGFI-B induces apoptosis in a number of cell lineages exposed to proapoptotic stimuli by directly targeting the mitochondria, inducing cytochrome c release. The present study was designed to determine the role of NGFI-B in cardiomyocytes of restraint-stressed rats. The NGFI-B content was increased in mitochondria and reduced in plasma as apoptosis increased. Analysis showed that NGFI-B induces cardiomyocyte apoptosis in restraint-stressed rats by mediating mitochondrial energy metabolism disorder. Several novel mitochondrial proteins, which correlate with NGFI-B, were reported in cardiomyocyte apoptosis of restraint-stressed rats. Five proteins associated with NGFI-B participate directly in mitochondrial energy metabolism. Studies of mitochondrial respiratory efficiency and ATP synthase activity strongly support the findings. These results provide significant information for comprehensively understanding the cellular mechanism of cardiovascular diseases. PMID:19412742

  5. Recent developments for an active UF6 gas target for photon-induced fission experiments

    NASA Astrophysics Data System (ADS)

    Freudenberger, M.; Eckardt, C.; Enders, J.; Göök, A.; von Neumann-Cosel, P.; Oberstedt, A.; Oberstedt, S.

    2013-12-01

    Recent developments for an active uranium-hexafluoride-loaded gas target as well as results on the detector gas properties are presented. The gas of choice is a mixture of argon with small amounts of UF6. This contribution presents the experimental setup and focusses on the electron drift velocity with increasing UF6 content. A time-dependent decrease in electron drift velocity is observed in our setup.

  6. Load-induced changes in older individual's hand-finger tremor are ameliorated with targeting.

    PubMed

    Kavanagh, Justin J; Cross, Troy J; Newell, Karl M; Morrison, Steven

    2014-04-15

    The purpose of this study was to investigate hand-finger tremor dynamics when a load was applied to the finger in a group of healthy older adults. Moreover, we sought to determine if projecting a representation of the subject's finger tremor on a target was capable of overcoming the effects of loading so that hand-finger interactions returned to a state that was similar to normal tremor. Eight healthy older males (67 ± 1 year) performed a postural pointing task, where tremor was assessed using lightweight accelerometers attached to the hand and finger. Tremor was then assessed when a laser pointer was attached to the finger and switched off (the load), and then with the laser pointer attached and switched on pointing at targets of 40 mm and 20mm in diameter. The main findings of this study were that 1) loading the finger resulted in a reduction in finger tremor amplitude and increased finger tremor regularity, but no change in hand tremor, 2) loading caused increased hand-finger 8-12 Hz cross wavelet coherence and phase synchrony, and 3) pointing at different targets while the finger was loaded resulted in an increase in finger tremor amplitude, and changes in inter-segmental coupling to the extent that hand-finger dynamics reflected normal unloaded conditions. Overall, these results illustrate that the damping effects of limb loading can be offset, in part, by altering the accuracy demands of the task to make the pointing action more challenging. PMID:24503237

  7. Colorimetric detection of gene transcript by target-induced three-way junction formation.

    PubMed

    Wang, Xuchu; Liu, Weiwei; Yin, Binbin; Yu, Pan; Duan, Xiuzhi; Liao, Zhaoping; Liu, Chunhua; Sang, Yiwen; Zhang, Gong; Chen, Yuhua; Tao, Zhihua

    2016-09-01

    Gene transcript often varies by alternative splicing, which plays different biological role that results in diversity of gene expression. Therefore, a simple and accurate identification of targeted transcript variant is of prime importance to achieve a precise molecular diagnosis. In this work, we presented a three-way junction based system where two split G-quadruplex forming sequences were coupled into two probes. Only upon the introduction of target gene transcript that offering a specific recognizable splicing site did the two probes assembled into three way junction conformation in a devised process, thus providing a functional G-quadruplex conformation that greatly enhanced hemin peroxidation. A notable resolution for gene splicing site detection was achieved. The detection limitation by colorimetric assay was 0.063μM, and this system has been proved to discriminate even in a single base false level around splicing site (about 3 times of single mismatched analyte to gain an equal signal by perfect analyte ). Furthermore, recoveries of 78.1%, 88.1%, 104.6% were obtained with 0.75μM, 0.25μM, 0.083μM of target, respectively, showing a capacity to further exploit a simple equipped device for gene transcript detection. PMID:27343570

  8. Measurement of proton-induced target fragmentation cross sections in carbon

    NASA Astrophysics Data System (ADS)

    Matsushita, K.; Nishio, T.; Tanaka, S.; Tsuneda, M.; Sugiura, A.; Ieki, K.

    2016-02-01

    In proton therapy, positron emitter nuclei are generated via the target nuclear fragmentation reactions between irradiated proton and nuclei constituting a human body. The proton-irradiated volume can be confirmed with measurement of annihilation γ-rays from the generated positron emitter nuclei. To achieve the high accuracy of proton therapy, in vivo dosimetry, i.e., evaluation of the irradiated dose during the treatment is important. To convert the measured activity distribution to irradiated dose, cross-sectional data for positron emitter production is necessary, which is currently insufficient in the treatment area. The purpose of this study is to collect cross-sectional data of 12C (p , pn)11C and 12C (p , p 2 n)10C reactions between the incident proton and carbon nuclei, which are important target nuclear fragmentation reactions, to estimate the range and exposure dose distribution in the patient's body. Using planar-type PET capable of measuring annihilation γ-rays at high positional resolution and thick polyethylene target, we measured cross-sectional data in continuous wide energy range. The cross section of 12C (p , pn)11C is in good agreement with existing experimental data. The cross section of 12C (p , p 2 n)10C is reported for the first data in the low-energy range of 67.6-10.5 MeV near the Bragg peak of proton beam.

  9. Targeting hepatitis B virus and human papillomavirus induced carcinogenesis: novel patented therapeutics.

    PubMed

    Kanwar, Rupinder K; Singh, Neha; Gurudevan, Sneha; Kanwar, Jagat R

    2011-05-01

    Viral infections leading to carcinogenesis tops the risk factors list for the development of human cancer. The decades of research has provided ample scientific evidence that directly links 10-15% of the worldwide incidence of human cancers to the infections with seven human viruses. Moreover, the insights gained into the molecular pathogenetic and immune mechanisms of hepatitis B virus (HBV) and human papillomavirus (HPV) viral transmission to tumour progression, and the identification of their viral surface antigens as well as oncoproteins have provided the scientific community with opportunities to target these virus infections through the development of prophylactic vaccines and antiviral therapeutics. The preventive vaccination programmes targeting HBV and high risk HPV infections, linked to hepatocellular carcinoma (HCC) and cervical cancer respectively have been recently reported to alter age-old cancer patterns on an international scale. In this review, with an emphasis on HBV and HPV mediated carcinogenesis because of the similarities and differences in their global incidence patterns, viral transmission, mortality, molecular pathogenesis and prevention, we focus on the development of recently identified HBV and HPV targeting innovative strategies resulting in several patents and patent applications. PMID:21517743

  10. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium

    PubMed Central

    Geldart, Kathryn; Borrero, Juan

    2015-01-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  11. Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium.

    PubMed

    Geldart, Kathryn; Borrero, Juan; Kaznessis, Yiannis N

    2015-06-01

    Antibiotic-resistant enterococcal infections are a major concern in hospitals where patients with compromised immunity are readily infected. Enterococcus faecium bacteria are of particular interest as these pathogens account for over 80% of vancomycin-resistant enterococcal infections. Antimicrobial peptides (AMPs) produced at the site of infection by engineered bacteria may offer a potential alternative to traditional antibiotics for the treatment of resistant bacteria such as E. faecium. For this mode of delivery to be effective, it is essential to identify a suitable protein expression system that can be used in the desired delivery bacterium. In this study, we describe a promising chloride-inducible promoter and its application in the bacterial delivery of AMPs from Lactococcus lactis to reduce counts of E. faecium bacteria in vitro. Reporter gene studies show that at chloride concentrations found within the human intestines, the chloride-inducible promoter exhibits high levels of protein expression compared to those of the commonly used nisin-inducible promoter. These results indicate that this system is powerful and would not require the exogenous administration of an inducer molecule. In its application for AMP production against E. faecium in vitro, L. lactis producing AMPs under the chloride promoter rapidly decreased E. faecium counts by nearly 10,000-fold. As an extension of this application, we also demonstrate the potential in using this type of delivery system in combination with traditional antibiotics to slow the development of resistance. Collectively, this study shows the promise of using a chloride-inducible promoter for the bacterial delivery of AMPs in the body for the treatment of vancomycin-resistant enterococci (VRE) and other antibiotic-resistant bacteria. PMID:25841002

  12. Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.

    PubMed

    Balakrishnan, Mini; Yant, Stephen R; Tsai, Luong; O'Sullivan, Christopher; Bam, Rujuta A; Tsai, Angela; Niedziela-Majka, Anita; Stray, Kirsten M; Sakowicz, Roman; Cihlar, Tomas

    2013-01-01

    HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly. PMID:24040198

  13. Non-Catalytic Site HIV-1 Integrase Inhibitors Disrupt Core Maturation and Induce a Reverse Transcription Block in Target Cells

    PubMed Central

    Tsai, Luong; O’Sullivan, Christopher; Bam, Rujuta A.; Tsai, Angela; Niedziela-Majka, Anita; Stray, Kirsten M.; Sakowicz, Roman; Cihlar, Tomas

    2013-01-01

    HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly. PMID:24040198

  14. Neutron yields for reactions induced by 120 GeV protons on thick copper target

    SciTech Connect

    Kajimoto, Tsuyoshi; Sanami, Toshiya; Iwamoto, Yosuke; Shigyo, Nobuhiro; Hagiwara, Masayuki; Saitoh, Kiwamu; Nakashima, Hiroshi; Ishibashi, Kenji; Lee, Hee-Seock; Ramberg, Eric; Coleman, Richard; /Fermilab

    2011-02-01

    We developed an experimental method to measure neutron energy spectrum for 120-GeV protons on a thick copper target at Fermilab Test Beam Facility (FTBF). The spectrum in the energy range from 16 to 1600 MeV was obtained for 60-cm long copper target by time-of-flight technique with an NE213 scintillator and 5.5-m flight path. Energy spectra of neutrons generated from an interaction with beam and materials are important to design shielding structure of high energy accelerators. Until now, the energy spectra for the incident energy up to 3 GeV have been measured by several groups, Ishibashi et al., Amian et al., and Leray et al. In the energy region above 3 GeV, few experimental data are available because of small number of facilities for neutron experiment. On the other hand, concerning simulation codes, theoretical models for particle generation and transportation are switched from intermediate to high energy one around this energy. The spectra calculated by the codes have not been examined using experimental data. In shielding experiments using 120 GeV hadron beam, experimental data shows systematic differences from calculations. Hagiwara et al. have measured leakage neutron spectra behind iron and concrete shield from 120 GeV proton on target at anti-proton target station in Fermilab by using Bonner Spheres with unfolding technique. In CERN, Nakao et al reported experimental results of neutron spectra behind iron and concrete wall from 120 GeV/c proton and pion mixed beam on copper by using NE213 liquid scintillators with unfolding technique. Both of the results reported systematic discrepancies between experimental and calculation results. Therefore, experimental data are highly required to verify neutron production part of calculations. In this study, we developed an experimental method to measure neutron energy spectrum for 120 GeV proton on target. The neutron energy was determined using time-of-flight technique. We used the Fermilab Test Beam Facility (FTBF

  15. Keap1 Cysteine 288 as a Potential Target for Diallyl Trisulfide-Induced Nrf2 Activation

    PubMed Central

    Kim, Sanghyun; Lee, Hee-Geum; Park, Sin-Aye; Kundu, Joydeb Kumar; Keum, Young-Sam; Cha, Young-Nam; Na, Hye-Kyung; Surh, Young-Joon

    2014-01-01

    Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its

  16. Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells*

    PubMed Central

    Kim, Seok-Jo; Cheresh, Paul; Williams, David; Cheng, Yuan; Ridge, Karen; Schumacker, Paul T.; Weitzman, Sigmund; Bohr, Vilhelm A.; Kamp, David W.

    2014-01-01

    Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity. PMID:24429287

  17. Carbohydrate-based inducers of cellular stress for targeting cancer cells.

    PubMed

    Ndombera, Fidelis T; VanHecke, Garrett C; Nagi, Shima; Ahn, Young-Hoon

    2016-03-01

    Small molecules that block the altered metabolism in cancer or increase the production of reactive oxygen species (ROS) are emerging as potential anti-cancer agents. Considering that various carbohydrates can be used for cellular energetics or protein N-glycosylation of which interruption can lead to cellular stress, we have synthesized and evaluated a library of N-aryl glycosides for induction of ROS and cytotoxicity in H1299 cancer cell line. Two N-aryl glycosides (K8 and H8) were identified that induce about 2-fold induction of ROS and cytotoxicity in H1299 cells. We further showed that the acetylated form of K8 (K8A) activates AMPK, and stabilizes p53 in HEK293 cells, and induce a higher cytotoxicity than 2-deoxy-d-glucose in H1299 cell line. PMID:26832785

  18. A Dual-Targeting, p53-Independent, Apoptosis-Inducing Platinum(II) Anticancer Complex, [Pt(BDIQQ)]Cl

    PubMed Central

    Suntharalingam, Kogularamanan; Wilson, Justin J.; Lin, Wei; Lippard, Stephen J.

    2014-01-01

    The therapeutic index and cellular mechanism of action of [Pt(BDIQQ)]Cl, a monocationic, square-planar platinum(II) complex, are reported. [Pt(BDIQQ)]Cl was used to treat several cell lines, including wild type and cisplatin-resistant ovarian carcinoma cells (A2780 and A2780CP70) and non-proliferating lung carcinoma cells (A549). [Pt(BDIQQ)]Cl selectively kills cancer over healthy cells and exhibits no cross-resistance with cisplatin. The mechanism of cell killing was established through detailed cell-based assays. [Pt(BDIQQ)]Cl exhibits dual-threat capabilities, targeting nuclear DNA and mitochondria simultaneously. [Pt(BDIQQ)]Cl induces DNA damage, leading to p53 enrichment, mitochondrial membrane potential depolarisation, and caspase-mediated apoptosis. [Pt(BDIQQ)]Cl also accumulates in the mitochondria, resulting in direct mitochondrial damage. Flow cytometric studies demonstrated that [Pt(BDIQQ)]Cl has no significant effect on cell cycle progression. Remarkably, p53-status is a not a determinant of [Pt(BDIQQ)]Cl activity. In p53-null cells, [Pt(BDIQQ)]Cl induces cell death through mitochondrial dysfunction. Cancers with p53-null status could therefore be targeted using [Pt(BDIQQ)]Cl. PMID:24514456

  19. Target induced interfacial self-assembly of nanoparticles: A new platform for reproducible quantification of copper ions.

    PubMed

    Yan, Ling; Zhang, Kun; Xu, Huiying; Ji, Ji; Wang, Yuning; Liu, Baohong; Yang, Pengyuan

    2016-09-01

    One of the main problems of the nanoparticle dispersion state change (e.g. from dispersion to aggregation) based surface-enhanced Raman scattering (SERS) detection is that the dynamic process of such in-solution reactions is always uncontrollable. This leads to poor reproducibility from a narrow time window of all such strategies, and finally great difference between the data from the diverse methods, and even between various sample batches. To address such problem, a facile, rapid SERS quantification protocol has been developed relying on target induced nanoparticle self-assembly at oil/water interfaces for copper ions analysis. In response to copper, the core-molecule-shell (CMS) nanoparticles spontaneously migrate to the interface and are assembled into densely packed arrays generating strong plasmonic coupling, which enables stable, sensitive and selective Raman quantitation, as well as visual detection. Also, this strategy shows capability for determination of large scale samples as the products can be stable for at least three weeks, and has been successfully applied to real sample detection. The developed Target Induced Nanoparticle Self-Assembled Interface (TINSAI) can be employed to both visual test and Raman quantitative detection, which would provide a platform for on-site screening as well as high-throughput detection with high sensitivity and selectivity. PMID:27343603

  20. Telocytes as potential targets in a cyclophosphamide-induced animal model of premature ovarian failure.

    PubMed

    Liu, Te; Wang, Suwei; Li, Qiong; Huang, Yongyi; Chen, Chuan; Zheng, Jin

    2016-09-01

    Premature ovarian failure (POF) refers to the presence of ovarian atrophic permanent amenorrhea in women under the age of 40. The pathogenesis of POF remains to be fully elucidated. Telocytes are a group of specialized cells with a small cell volume and very long cytoplasmic prolongations with dichotomous branching. Previous studies have indicated that telocytes function to support the trachea and serve as stem cell niches. Although it has been confirmed that telocytes are present in numerous organs in mammals, it remains to be determined whether they are present in ovarian tissues and whether they are involved in the development of POF. The present study used a cyclophosphamide-induced mouse model of POF. Hematoxylin and eosin staining and an enzyme‑linked immunosorbent assay revealed that cyclophosphamide induced edema and apoptosis of ovarian stromal and granulosa cells and increased atretic follicles. In addition, cyclophosphamide induced abnormal peripheral blood FSH and E2 levels in mice. Transmission electron microscopy revealed a small number of telocyte‑like cell structures in the ovarian stroma of wild‑type mice. In addition, flow cytometry and immunohistochemical staining results suggested that the number of cluster of differentiation (CD)34/platelet‑derived growth factor receptor (PDGFR)α, CD34/PDGFRβ and CD34/vimentin double‑positive cells in the ovaries of POF mice was significantly decreased compared with wild‑type mice. In conclusion, mouse ovarian tissues appear to contain telocytes, and cyclophosphamide treatment significantly reduced the number of ovarian telocytes. Therefore, telocytes may serve as a potential novel marker of POF induced by cyclophosphamide. PMID:27485835

  1. Screening for biomarkers of liver injury induced by Polygonum multiflorum: a targeted metabolomic study

    PubMed Central

    Dong, Qin; Li, Na; Li, Qi; Zhang, Cong-En; Feng, Wu-Wen; Li, Guang-Quan; Li, Rui-Yu; Tu, Can; Han, Xue; Bai, Zhao-Fang; Zhang, Ya-Ming; Niu, Ming; Ma, Zhi-Jie; Xiao, Xiao-He; Wang, Jia-Bo

    2015-01-01

    Heshouwu (HSW), the dry roots of Polygonum multiflorum, a classical traditional Chinese medicine is used as a tonic for a wide range of conditions, particularly those associated with aging. However, it tends to be taken overdose or long term in these years, which has resulted in liver damage reported in many countries. In this study, the indicative roles of nine bile acids (BAs) were evaluated to offer potential biomarkers for HSW induced liver injury. Nine BAs including cholic acid (CA) and chenodeoxycholic acid (CDCA), taurocholic acid (TCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA) in rat bile and serum were detected by a developed LC-MS method after 42 days treatment. Partial least square-discriminate analysis (PLS-DA) was applied to evaluate the indicative roles of the nine BAs, and metabolism of the nine BAs was summarized. Significant change was observed for the concentrations of nine BAs in treatment groups compared with normal control; In the PLS-DA plots of nine BAs in bile, normal control and raw HSW groups were separately clustered and could be clearly distinguished, GDCA was selected as the distinguished components for raw HSW overdose treatment group. In the PLS-DA plots of nine BAs in serum, the normal control and raw HSW overdose treatment group were separately clustered and could be clearly distinguished, and HDCA was selected as the distinguished components for raw HSW overdose treatment group. The results indicated the perturbation of nine BAs was associated with HSW induced liver injury; GDCA in bile, as well as HDCA in serum could be selected as potential biomarkers for HSW induced liver injury; it also laid the foundation for the further search on the mechanisms of liver injury induced by HSW. PMID:26483689

  2. Screening for biomarkers of liver injury induced by Polygonum multiflorum: a targeted metabolomic study.

    PubMed

    Dong, Qin; Li, Na; Li, Qi; Zhang, Cong-En; Feng, Wu-Wen; Li, Guang-Quan; Li, Rui-Yu; Tu, Can; Han, Xue; Bai, Zhao-Fang; Zhang, Ya-Ming; Niu, Ming; Ma, Zhi-Jie; Xiao, Xiao-He; Wang, Jia-Bo

    2015-01-01

    Heshouwu (HSW), the dry roots of Polygonum multiflorum, a classical traditional Chinese medicine is used as a tonic for a wide range of conditions, particularly those associated with aging. However, it tends to be taken overdose or long term in these years, which has resulted in liver damage reported in many countries. In this study, the indicative roles of nine bile acids (BAs) were evaluated to offer potential biomarkers for HSW induced liver injury. Nine BAs including cholic acid (CA) and chenodeoxycholic acid (CDCA), taurocholic acid (TCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid (HDCA) in rat bile and serum were detected by a developed LC-MS method after 42 days treatment. Partial least square-discriminate analysis (PLS-DA) was applied to evaluate the indicative roles of the nine BAs, and metabolism of the nine BAs was summarized. Significant change was observed for the concentrations of nine BAs in treatment groups compared with normal control; In the PLS-DA plots of nine BAs in bile, normal control and raw HSW groups were separately clustered and could be clearly distinguished, GDCA was selected as the distinguished components for raw HSW overdose treatment group. In the PLS-DA plots of nine BAs in serum, the normal control and raw HSW overdose treatment group were separately clustered and could be clearly distinguished, and HDCA was selected as the distinguished components for raw HSW overdose treatment group. The results indicated the perturbation of nine BAs was associated with HSW induced liver injury; GDCA in bile, as well as HDCA in serum could be selected as potential biomarkers for HSW induced liver injury; it also laid the foundation for the further search on the mechanisms of liver injury induced by HSW. PMID:26483689

  3. Suppression of tumor growth by designed dimeric epidithiodiketopiperazine targeting hypoxia-inducible transcription factor complex.

    PubMed

    Dubey, Ramin; Levin, Michael D; Szabo, Lajos Z; Laszlo, Csaba F; Kushal, Swati; Singh, Jason B; Oh, Philip; Schnitzer, Jan E; Olenyuk, Bogdan Z

    2013-03-20

    Hypoxia is a hallmark of solid tumors, is associated with local invasion, metastatic spread, resistance to chemo- and radiotherapy, and is an independent, negative prognostic factor for a diverse range of malignant neoplasms. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, altered energy metabolism, antiapoptotic, and pro-proliferative mechanisms that promote tumor growth. A designed transcriptional antagonist, dimeric epidithiodiketopiperazine (ETP 2), selectively disrupts the interaction of HIF1α with p300/CBP coactivators and downregulates the expression of hypoxia-inducible genes. ETP 2 was synthesized via a novel homo-oxidative coupling of the aliphatic primary carbons of the dithioacetal precursor. It effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of cell lines representing breast and lung cancers. We observed an outstanding antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. Treatment with either form of ETP 2 (1 mg/kg) resulted in a rapid regression of tumor growth that lasted for up to 14 days. These results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance. PMID:23448368

  4. Telocytes as potential targets in a cyclophosphamide-induced animal model of premature ovarian failure

    PubMed Central

    Liu, Te; Wang, Suwei; Li, Qiong; Huang, Yongyi; Chen, Chuan; Zheng, Jin

    2016-01-01

    Premature ovarian failure (POF) refers to the presence of ovarian atrophic permanent amenorrhea in women under the age of 40. The pathogenesis of POF remains to be fully elucidated. Telocytes are a group of specialized cells with a small cell volume and very long cytoplasmic prolongations with dichotomous branching. Previous studies have indicated that telocytes function to support the trachea and serve as stem cell niches. Although it has been confirmed that telocytes are present in numerous organs in mammals, it remains to be determined whether they are present in ovarian tissues and whether they are involved in the development of POF. The present study used a cyclophosphamide-induced mouse model of POF. Hematoxylin and eosin staining and an enzyme-linked immunosorbent assay revealed that cyclophosphamide induced edema and apoptosis of ovarian stromal and granulosa cells and increased atretic follicles. In addition, cyclophosphamide induced abnormal peripheral blood FSH and E2 levels in mice. Transmission electron microscopy revealed a small number of telocyte-like cell structures in the ovarian stroma of wild-type mice. In addition, flow cytometry and immunohistochemical staining results suggested that the number of cluster of differentiation (CD)34/platelet-derived growth factor receptor (PDGFR)α, CD34/PDGFRβ and CD34/vimentin double-positive cells in the ovaries of POF mice was significantly decreased compared with wild-type mice. In conclusion, mouse ovarian tissues appear to contain telocytes, and cyclophosphamide treatment significantly reduced the number of ovarian telocytes. Therefore, telocytes may serve as a potential novel marker of POF induced by cyclophosphamide. PMID:27485835

  5. Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  6. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    PubMed

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  7. Intracellular monitoring of target protein production in Staphylococcus aureus by peptide tag‐induced reporter fluorescence

    PubMed Central

    Gauger, Tina; Weihs, Felix; Mayer, Sonja; Krismer, Bernhard; Liese, Jan; Kull, Melanie; Bertram, Ralph

    2012-01-01

    Summary An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time‐ and concentration‐dependent production of mCherry could be correlated to TetR‐controlled GFPmut2 activity. This approach can potentially be extended to native S. aureus proteins. PMID:21958360

  8. Sensitization of acute lymphoblastic leukemia cells for LCL161-induced cell death by targeting redox homeostasis.

    PubMed

    Haß, Christina; Belz, Katharina; Schoeneberger, Hannah; Fulda, Simone

    2016-04-01

    Disturbed redox homeostasis with both elevated reactive oxygen species (ROS) levels and antioxidant defense mechanisms has been reported in acute lymphoblastic leukemia (ALL). We therefore hypothesized that inhibition of pathways responsible for ROS detoxification renders ALL cells more susceptible for cell death. Here, we report that pharmacological inhibitors of key pathways for the elimination of ROS, i.e. Erastin, buthionine sulfoximine (BSO) and Auranofin, sensitize ALL cells for cell death upon treatment with the Smac mimetic LCL161 that antagonizes Inhibitor of Apoptosis (IAP) proteins. Erastin, BSO or Auranofin significantly increase LCL161-induced cell death and also act in concert with LCL161 to profoundly suppress long-term clonogenic survival in several ALL cell lines. Erastin or BSO cooperates with LCL161 to stimulate ROS production and lipid peroxidation prior to cell death. ROS production and lipid peroxidation are required for this cotreatment-induced cell death, since ROS scavengers or pharmacological inhibition of lipid peroxidation provides significant protection against cell death. These results emphasize that inhibition of antioxidant defense mechanisms can serve as a potent approach to prime ALL cells for LCL161-induced cell death. PMID:26774450

  9. Myricetin Selectively Induces Apoptosis on Cancerous Hepatocytes by Directly Targeting Their Mitochondria.

    PubMed

    Seydi, Enayatollah; Rasekh, Hamid Reza; Salimi, Ahmad; Mohsenifar, Zhaleh; Pourahmad, Jalal

    2016-09-01

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death. In patients for whom HCC could not be detected early, current treatments show poor tolerance and low efficacy. So, alternative therapies with good efficacy are urgently needed. The aim of this research was to evaluate the selective apoptotic effects of myricetin (MYR), a flavonoid compound, on hepatocytes and mitochondria obtained from the liver of HCC rats. In this study, HCC induced by diethylnitrosamine (DEN), as an initiator, and 2-acetylaminofluorene (2-AAF), as a promoter. To confirm the HCC induction, serum levels of alpha-fetoprotein (AFP), AST, AST and ALP and histopathological changes in the liver tissue were evaluated. Rat liver hepatocytes and mitochondria for evaluation of the selective cytotoxic effects of MYR were isolated, and mitochondrial and cellular parameters related to apoptosis signalling were then determined. Our results showed that MYR was able to induce cytotoxicity only in hepatocytes from the HCC but not from the untreated control group. Besides, MYR (12.5, 25 and 50 μM) induced a considerable increase in reactive oxygen species (ROS) level, mitochondrial swelling, mitochondrial membrane permeabilization (MMP) and cytochrome c release only in cancerous but not in untreated normal hepatocyte mitochondria. MYR selectively increased caspase-3 activation and apoptotic phenotypes in HCC, but not untreated normal hepatocytes. Finally, our finding underlines MYR as a promising therapeutic candidate against HCC and recommends the compound for further studies. PMID:26919160

  10. Silkworm Thorn Stem Extract Targets RSK2 and Suppresses Solar UV-Induced Cyclooxygenase-2 Expression

    PubMed Central

    Kim, Jong-Eun; Lee, Ki Won

    2015-01-01

    Excessive exposure to solar UV (sUV) is associated with numerous human skin disorders, such as carcinogenesis, skin photoaging and skin inflammation. Silkworm Thorn (Cudraniatricuspidata, SW) is a plant belonging to the Moraceae family and widely present throughout Korea, China, and Japan. Most parts of the tree (including the fruit, leaf, stem, root, and bark) is consumable as a functional food or tea. In this study, we found that SW extract (SWE) inhibited the elevated expression of sUV-induced cyclooxygenase (COX)-2 levels in both HaCaT and JB6 cells. Levels of nuclear factor-κB and activator protein-1, two crucial transcription factors involved in COX-2 expression, were elevated by sUV treatment. Treatment with SWE abolished this activation. SWE also inhibited sUV-induced histone H3 phosphorylation. However, sUV-induced phosphorylation of Akt, c-Jun N-terminal kinase and p38 kinase remained unchanged in the presence of SWE. SWE inhibited RSK2 activity, and pull-down assays using SWE-Sepharose beads revealed that SWE binds directly with RSK2 in an ATP-competitive manner. These results suggest a potential for SWE to be developed as a cosmeceutical material and functional food constituent for the promotion of skin health. PMID:26506342

  11. MiR-133b ameliorates axon degeneration induced by MPP(+) via targeting RhoA.

    PubMed

    Niu, M; Xu, R; Wang, J; Hou, B; Xie, A

    2016-06-14

    Increasing evidence suggests that microRNAs (miRs) play a significant role in the pathogenesis of Parkinson's disease (PD). MiR-133b, which is significantly decreased in the PD midbrain, has recently been shown to promote neurite outgrowth and enhance neural functional recovery. However, the role of miR-133b in PD has not been clearly established. Here, using a well-established PD model culture based on the neurotoxin 1-methyl-4-phenyl-pyridinium (MPP(+)), we demonstrated that miR-133b could promote axon outgrowth in dopaminergic neurons (DNs) and ameliorated MPP(+)-induced axon degeneration. Additional experiments suggested that the mechanisms of this miR-133b-mediated effect might rely on RhoA inhibition. We demonstrated that RhoA, an inhibitor of axonal growth, was increased in DNs under MPP(+) treatment, and this increase could be attenuated by miR-133b overexpression. Moreover, we demonstrated that the induced expression of miR-133b could inhibit α-synuclein, which is critically involved in the pathological process of PD. Furthermore, we found that overexpression of miR-133b abrogated the MPP(+)-induced decrease in the Bcl-2/Bax ratio and upregulated phosphorylated Akt (p-Akt), which is a pro-survival kinase. Together these findings reveal novel roles for miR-133b in the pathogenesis of PD and provide new therapeutic avenues for the treatment of the disease. PMID:27012608

  12. Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma

    PubMed Central

    Terragna, Carolina; Martello, Marina; Dico, Angela F.; Solaini, Giancarlo; Baracca, Alessandra; Sgarbi, Gianluca; Pasquinelli, Gianandrea; Valente, Sabrina; Zamagni, Elena; Tacchetti, Paola; Martinelli, Giovanni; Cavo, Michele

    2014-01-01

    The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment. PMID:24732040

  13. Hypoxia Inducible Factor 1 as a Therapeutic Target in Ischemic Stroke

    PubMed Central

    Shi, H

    2010-01-01

    In stroke research, a significant focus is to develop therapeutic strategies that prevent neuronal death and improve recovery. Yet, few successful therapeutic strategies have emerged. Hypoxia-inducible factor 1 (HIF-1) is a key regulator in hypoxia. It has been suggested to be an important player in neurological outcomes following ischemic stroke due to the functions of its downstream genes. These include genes that promote glucose metabolism, angiogenesis, erythropoiesis, and cell survival. Many lines of evidence have shown that HIF-1 is induced in ischemic brains. Importantly, it seems that HIF-1 is primarily induced in the salvageable tissue of an ischemic brain, penumbra. However, the effect of HIF-1 on neuronal tissue injuries is still debatable based on evidence from in vitro and preclinical studies. Furthermore, it is of importance to understand the mechanism of HIF-1 degradation after its induction in ischemic brain. This review provides a present understanding of the mechanism of HIF-1 induction in ischemic neurons and the potential effect of HIF-1 on ischemic brain tissue. The author also elaborates on potential therapeutic approaches through understanding of the induction mechanism and of the potential role of HIF-1 in ischemic stroke. PMID:19903149

  14. Silibinin attenuates methotrexate-induced pulmonary injury by targeting oxidative stress

    PubMed Central

    KALEMCI, SERDAR; TOPAL, YASAR; CELIK, SERKAN YASAR; YILMAZ, NIGAR; BEYDILLI, HALIL; KOSAR, MEHMET ILKAY; DIRICAN, NIGAR; ALTUNTAS, IRFAN

    2015-01-01

    The aim of the present study was to assess the protective effect of silibinin against methotrexate (MTX)-induced pulmonary toxicity. Rats were divided into four groups (MTX, MTX + silibinin, silibinin and control. MTX was injected intraperitoneally (i.p) into female Wistar rats (10 mg/kg/day for 3 days), which resulted in significant increases in the serum levels of alanine aminotransferase, aspartate aminotransferase and oxidant enzymes, including nitric oxide and myeloperoxidase. Furthermore, significant reductions were detected in the serum activity levels of the antioxidative enzymes, glutathione peroxidase and superoxide dismutase, when compared with the control group. However, administration of silibinin (100 mg/kg/day for 10 days, i.p.) was shown to ameliorate the MTX-induced pulmonary toxicity, as indicated by the normalization of the oxidative stress parameters. Furthermore, silibinin treatment was demonstrated to reduce the histopathological changes associated with MTX. In conclusion, silibinin exhibited protective effects against MTX-induced pulmonary toxicity, which may be attributed to its antioxidant activity. PMID:26622344

  15. Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria.

    PubMed

    Weidner, Christopher; Rousseau, Morten; Micikas, Robert J; Fischer, Cornelius; Plauth, Annabell; Wowro, Sylvia J; Siems, Karsten; Hetterling, Gregor; Kliem, Magdalena; Schroeder, Frank C; Sauer, Sascha

    2016-01-22

    A known (1) and a structurally related new natural product (2), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of Glycyrrhiza foetida. Compound 1 (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound 2 (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC50 values ranging from 8 to 16 μM in these cancer cell lines). Notably, amorfrutin C (2) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound 2 induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that 2 disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC50 0.6 μM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure-activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of 2 as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (2) represents a promising lead for the development of anticancer drugs. PMID:26731300

  16. Emerging Mechanistic Targets in Lung Injury Induced by Combustion-Generated Particles

    PubMed Central

    Fariss, Marc W.; Gilmour, M. Ian; Reilly, Christopher A.; Liedtke, Wolfgang; Ghio, Andrew J.

    2013-01-01

    The mechanism for biological effect following exposure to combustion-generated particles is incompletely defined. The identification of pathways regulating the acute toxicological effects of these particles provides specific targets for therapeutic manipulation in an attempt to impact disease following exposures. Transient receptor potential (TRP) cation channels were identified as “particle sensors” in that their activation was coupled with the initiation of protective responses limiting airway deposition and inflammatory responses, which promote degradation and clearance of the particles. TRPA1, V1, V4, and M8 have a capacity to mediate adverse effects after exposure to combustion-generated particulate matter (PM); relative contributions of each depend upon particle composition, dose, and deposition. Exposure of human bronchial epithelial cells to an organic extract of diesel exhaust particle was followed by TRPV4 mediating Ca++ influx, increased RAS expression, mitogen-activated protein kinase signaling, and matrix metalloproteinase-1 activation. These novel pathways of biological effect can be targeted by compounds that specifically inhibit critical signaling reactions. In addition to TRPs and calcium biochemistry, humic-like substances (HLS) and cell/tissue iron equilibrium were identified as potential mechanistic targets in lung injury after particle exposure. In respiratory epithelial cells, iron sequestration by HLS in wood smoke particle (WSP) was associated with oxidant generation, cell signaling, transcription factor activation, and release of inflammatory mediators. Similar to WSP, cytotoxic insoluble nanosized spherical particles composed of HLS were isolated from cigarette smoke condensate. Therapies that promote bioelimination of HLS and prevent the disruption of iron homeostasis could function to reduce the harmful effects of combustion-generated PM exposure. PMID:23322347

  17. Nanosecond laser-induced shock propagation in and above organic liquid and solid targets

    NASA Astrophysics Data System (ADS)

    O'Malley, S. M.; Zinderman, B.; Schoeffling, J.; Jimenez, R.; Naddeo, J. J.; Bubb, D. M.

    2014-11-01

    The study of shock propagation in air and liquid can play an important role in understanding light-matter interactions during laser processing experiments. In this work, we perform plume shadowgraphy experiments on liquid and solid targets of acetone and toluene and calculate the velocity and pressure at the leading edge of the shock front. Our results are compared to recent work in which early blast wave dynamics are studied and the applicability of the classical Taylor-Sedov model is assessed for our data. We observe an enhanced vertical expansion in the shockwave that is attributable to absorption and heating above the surface.

  18. Molecular Mechanism and Potential Targets for Blocking HPV-Induced Lesion Development

    PubMed Central

    Guzmán-Olea, E.; Bermúdez-Morales, V. H.; Peralta-Zaragoza, O.; Torres-Poveda, K.; Madrid-Marina, V.

    2012-01-01

    Persistent infection with high-risk HPV is the etiologic agent associated with the development of cervical cancer (CC) development. However, environmental, social, epidemiological, genetic, and host factors may have a joint influence on the risk of disease progression. Cervical lesions caused by HPV infection can be removed naturally by the host immune response and only a small percentage may progress to cancer; thus, the immune response is essential for the control of precursor lesions and CC. We present a review of recent research on the molecular mechanisms that allow HPV-infected cells to evade immune surveillance and potential targets of molecular therapy to inhibit tumor immune escape. PMID:22220169

  19. Molecular Mechanism and Potential Targets for Blocking HPV-Induced Lesion Development.

    PubMed

    Guzmán-Olea, E; Bermúdez-Morales, V H; Peralta-Zaragoza, O; Torres-Poveda, K; Madrid-Marina, V

    2012-01-01

    Persistent infection with high-risk HPV is the etiologic agent associated with the development of cervical cancer (CC) development. However, environmental, social, epidemiological, genetic, and host factors may have a joint influence on the risk of disease progression. Cervical lesions caused by HPV infection can be removed naturally by the host immune response and only a small percentage may progress to cancer; thus, the immune response is essential for the control of precursor lesions and CC. We present a review of recent research on the molecular mechanisms that allow HPV-infected cells to evade immune surveillance and potential targets of molecular therapy to inhibit tumor immune escape. PMID:22220169

  20. Non-cell autonomous effects of targeting inducible PGE2 synthesis during inflammation-associated colon carcinogenesis

    PubMed Central

    Nakanishi, Masako; Perret, Christine; Meuillet, Emmanuelle J.; Rosenberg, Daniel W.

    2015-01-01

    Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc Δ14/+ in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc Δ14/+ mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc Δ14/+ mice as PGE2 contributes to the growth and expansion of the

  1. Non-cell autonomous effects of targeting inducible PGE2 synthesis during inflammation-associated colon carcinogenesis.

    PubMed

    Nakanishi, Masako; Perret, Christine; Meuillet, Emmanuelle J; Rosenberg, Daniel W

    2015-04-01

    Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc (Δ14/+) in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of

  2. Direct involvement of CD56 in cytokine-induced killer-mediated lysis of CD56+ hematopoietic target cells.

    PubMed

    Valgardsdottir, Rut; Capitanio, Cristina; Texido, Gemma; Pende, Daniela; Cantoni, Claudia; Pesenti, Enrico; Rambaldi, Alessandro; Golay, Josée; Introna, Martino

    2014-12-01

    Cytokine-induced killer (CIK) cells are in-vitro-expanded T lymphocytes that represent a heterogeneous population. A large majority of CIK cells are CD3(+)CD56(+), and this population has been shown to confer a cytotoxic effect against tumor targets. The scope of this work was to study whether CD56 has a direct role in CIK-mediated cytotoxicity. Blocking of CD56 with the anti-CD56 monoclonal antibody GPR165 significantly reduced CIK-mediated lysis of three CD56(+) hematopoietic tumor cell lines (AML-NS8, NB4, and KCL22), whereas no effect was observed on three CD56(-) hematopoietic tumor cell lines (K562, REH, and MOLT-4). Knockdown of CD56 in CIK cells by short interfering RNA made the cells less cytotoxic against a CD56(+) target, and knockdown of CD56 in target cells with lentiviral short hairpin RNA significantly altered their susceptibility to CIK-mediated lysis. Our data suggest that homophilic interaction between CD56 molecules may occur in tumor-cell recognition, leading to CIK-mediated cell death. PMID:25201755

  3. A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds.

    PubMed

    Silva, Isba; Real, Lilian J; Ward, Matthew S; Xu, H Howard

    2014-06-01

    One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens. PMID:24622888

  4. MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN

    PubMed Central

    Wang, Yun; Chen, Hong; Fu, Yonglun; Ai, Ai; Xue, Songguo; Lyu, Qifeng; Kuang, Yanping

    2013-01-01

    MiR-195, which exhibits a proliferation-inhibiting role in different tumors, has been reported to be down-regulated in the ectopic endometrium. The aim of this study was to determine the impact of miR-195 on the biological characteristic of the endometrial stromal cells (ESCs). MiR-195 has been presumed to target the 3’-untranslated regions (3’-UTR) of Fractalkine (FKN), which also plays important roles in endometriosis. Fluorescence reporter assays showed that miR-195 effectively binds to the 3’-UTR of FKN. The normal ESCs showed a significant higher miR-195 expression than that of eutopic and ectopic ESCs associated with endometriosis, while the FKN expression showed opposite results. MiR-195 mimics inhibited proliferation and growth and induced apoptosis of eutopic ESCs, and these effects were abolished by FKN-siRNA. miR-195 could decrease the expression of survivin, matrix metalloproteinase-9 (MMP9) and up-regulate the expression of CD82, tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 of eutopic ESCs by targeting FKN. Our study has demonstrated for the first time that miR-195 plays important roles in regulating the functions of ESCs through targeting FKN. The information may be useful for developing a new therapeutic strategy for endometriosis. PMID:24294368

  5. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

    PubMed

    Mamta; Reddy, K R K; Rajam, M V

    2016-02-01

    Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera. PMID:26659592

  6. MicroRNA-101 inhibits cell proliferation and induces apoptosis by targeting EYA1 in breast cancer.

    PubMed

    Guan, Haitao; Dai, Zhijun; Ma, Yuguang; Wang, Zhongwei; Liu, Xiaoxu; Wang, Xijing

    2016-06-01

    MicroRNAs (miRNAs or miRs) regulate gene expression by negatively modulating the stability or translational efficiency of their target genes by targeting the 3'-untranslated region (3'-UTR). Aberrant miRNA expression has been reported in various types of cancer; miRNAs can function as either oncogenes or tumor suppressor genes in cancer. In this study, we examined the expression level of miR‑101 in breast cancer tissues and cell lines by RT-qPCR, and found that miR‑101 expression was downregulated in breast cancer tissues and cell lines; indeed, in 6 of the 28 tissue samples, miR‑101 could not be detected. Furthermore, miR‑101, when transfected into SKBR3 cells, inhibited cell proliferation and promoted apoptosis, while miR‑101 inhibitor had the opposite effect. A dual-luciferase reporter assay revealed that miR‑101 targeted the 3'-UTR of eyes absent homolog 1 (Drosophila) (EYA1). Western blot analysis demonstrated a significantly decreased protein level of EYA1 in the SKBR3 cells transfected with miR‑101 mimic, whereas transfection with miR‑101 inhibitor led to an increased level of EYA1. Moreover, an increased expression of EYA1 was also found in breast cancer tissues and cell lines. The silencing of EYA1 using siRNA targeting EYA1 (EYA1‑siRNA) significantly inhibited SKBR3 cell proliferation and promoted apoptosis, and also suppressed the increased proliferation induced by transfection with miR‑101 inhibitor. The protein expression levels of Notch signaling components (jagged1, Hes1 and Hey1) were significantly decreased by transfection with miR‑101 mimic and EYA1-siRNA, and were increased by transfection with miR‑101 inhibitor. Furthermore, the elevated protein expression levels of jagged1, Hes1 and Hey1 induced by transfection with miR‑101 inhibitor in the SKBR3 cells were significantly decreased by transfection with EYA1-siRNA. Taken together, these results suggest that miR‑101 is down-regulated in breast cancer, and can

  7. [Study of non-visually induced smooth pursuit eye movements and their predictability using pseudorandom target motion].

    PubMed

    Watanabe, N; Hashiba, M

    1997-12-01

    It has been found that the smooth pursuit eye movements (SPEM) are elicited by not only visual stimuli but also non-visual information such as the subject's fingertip movement and a moving sound source. We have already reported the quantitative analysis of SPEM which were induced by somatosensory and acoustic information. In the previous study, we used a sinusoidal waveform that could be highly predictable. Since it is wellknown that predictive control has an important role in the normal SPEM, we expect the predictive control to function in non-visually induced SPEM (NVSPEM). We quantitatively analyzed NVSPEM and normal SPEM evoked by pseudorandom target motion in ten human subjects who had no ocular, oculomotor or vestibular disorders. NVSPEM were induced by the following two non-visual targets: 1, subjects' fingertip motion as a somatosensory target ("Somato"), 2, a small loudspeaker (3-cm diameter.) generating white noise with an intensity of about 60 dB (A) as an acoustic target ("Acoustic"). A servo-controlled swing arm of 50cm was used to drive the subject's fingertip and the acoustic target of the small loudspeaker. The horizontal motion of the swing arm was controlled by a personal computer. The pseudorandom target motion was generated by mixing four sinusoids (0.1, 0.2, 0.4, 0.8 Hz) of which the phases were randomly selected and the peak velocities were equally set at 19 deg/s. The mean peak velocity of the target was 26.2 deg/s and the amplitude was limited within 15 deg. Horizontal eye movements were recorded by DC electro-oculography and on an analogue datatape. The experiment was performed for 30 s in complete darkness so that the subjects' fingertip and loudspeaker as such remain invisible to the subject. Signals from the data recorder were smoothed by a low pass analogue filter of 20Hz, after digitization with a sampling frequency of 200 Hz and precision of 12 bits, and stored on a computer. The slow and quick eye movement components, both of which

  8. Overexpression of miR-34c inhibits high glucose-induced apoptosis in podocytes by targeting Notch signaling pathways

    PubMed Central

    Liu, Xiang-Dong; Zhang, Lian-Yun; Zhu, Tie-Chui; Zhang, Rui-Fang; Wang, Shu-Long; Bao, Yan

    2015-01-01

    Recent findings have shown that microRNAs play critical roles in the pathogenesis of diabetic nephropathy. miR-34c has been found to inhibit fibrosis and the epithelial-mesenchymal transition of kidney cells. However, the role of miR-34c in diabetic nephropathy has not been well studied. The current study was designed to investigate the role and potential underlying mechanism of miR-34c in regulating diabetic nephropathy. After treating podocytes with high glucose (HG) in vitro, we found that miR-34c was downregulated and that overexpression of miR-34c inhibited HG-induced podocyte apoptosis. The direct interaction between miR-34c and the 3’-untranslated region (UTR) of Notch1 and Jagged1 was validated by dual-luciferase reporter assay. Moreover, Notch1 and Jagged1 as putative targets of miR-34c were downregulated by miR-34c overexpression in HG-treated podocytes. Overexpression of miR-34c inhibited HG-induced Notch signaling pathway activation, as indicated by decreased expression of the Notch intracellular domain (NICD) and downstream genes including Hes1 and Hey1. Furthermore, miR-34c overexpression increased the expression of the anti-apoptotic gene Bcl-2, and decreased the expression of the pro-apoptotic protein Bax and cleaved Caspase-3. Additionally, the phosphorylation of p53 was also downregulated by miR-34c overexpression. Taken together, our findings suggest that miR-34c overexpression inhibits the Notch signaling pathway by targeting Notch1 and Jaggged1 in HG-treated podocytes, representing a novel and potential therapeutic target for the treatment of diabetic nephropathy. PMID:26191142

  9. Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

    SciTech Connect

    Brüning, Ansgar Matsingou, Christina; Brem, German Johannes; Rahmeh, Martina; Mylonas, Ioannis

    2012-10-15

    Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir. Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.

  10. Cyclophilin A as a New Therapeutic Target for Hepatitis C Virus-induced Hepatocellular Carcinoma

    PubMed Central

    2013-01-01

    Hepatocellular carcinoma (HCC) related to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is thought to account for more than 80% of primary liver cancers. Both HBV and HCV can establish chronic liver inflammatory infections, altering hepatocyte and liver physiology with potential liver disease progression and HCC development. Cyclophilin A (CypA) has been identified as an essential host factor for the HCV replication by physically interacting with the HCV non structural protein NS5A that in turn interacts with RNA-dependent RNA polymerase NS5B. CypA, a cytosolic binding protein of the immunosuppressive drug cyclosporine A, is overexpressed in many cancer types and often associated with malignant transformation. Therefore, CypA can be a good target for molecular cancer therapy. Because of antiviral activity, the CypA inhibitors have been tested for the treatment of chronic hepatitis C. Nonimmunosuppressive Cyp inhibitors such as NIM811, SCY-635, and Alisporivir have attracted more interests for appropriating CypA for antiviral chemotherapeutic target on HCV infection. This review describes CypA inhibitors as a potential HCC treatment tool that is contrived by their obstructing chronic HCV infection and summarizes roles of CypA in cancer development. PMID:24227937

  11. Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

    PubMed

    Wen, Qiang Jeremy; Yang, Qiong; Goldenson, Benjamin; Malinge, Sébastien; Lasho, Terra; Schneider, Rebekka K; Breyfogle, Lawrence J; Schultz, Rachael; Gilles, Laure; Koppikar, Priya; Abdel-Wahab, Omar; Pardanani, Animesh; Stein, Brady; Gurbuxani, Sandeep; Mullally, Ann; Levine, Ross L; Tefferi, Ayalew; Crispino, John D

    2015-12-01

    Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-β. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit. PMID:26569382