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61.
Marta J. Gonzalez-Hernandez Anupama Pal Kofi E. Gyan Marie-Eve Charbonneau Hollis D. Showalter Nicholas J. Donato Mary O'Riordan Christiane E. Wobus 《PloS one》2014,9(4)
Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility. 相似文献
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Acestor N Zíková A Dalley RA Anupama A Panigrahi AK Stuart KD 《Molecular & cellular proteomics : MCP》2011,10(9):M110.006908
The mitochondrial respiratory chain is comprised of four different protein complexes (I–IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by FoF1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.Mitochondria are dynamic organelles essential for cellular life, death, and differentiation of virtually every eukaryotic cell. They house systems for energy production through oxidative phosphorylation, synthesis of key metabolites, and iron-sulfur cluster assembly. The oxidative phoshorylation system of eukaryotic mitochondria comprises five major complexes located in the mitochondrial (mt)1 inner membrane, and often abbreviated as mt complexes I–V. The redox energy of the substrates NADH and succinate is first converted into an electrochemical proton potential across the inner mt membrane by respiratory complexes I (NADH:ubiquinone reductase), II (SDH, succinate:ubiquinone reductase), III (bc1, ubiquinone:cytochrome c reductase), and IV (cytochrome c oxidase). The electrochemical proton potential is then used by complex V (FoF1-ATP synthase) to synthesize ATP from ADP and inorganic phosphate, a mechanism that has essentially remained unchanged from bacteria to human (1). However, parasitic organisms have exploited unique energy metabolic pathways by adapting to their natural host habitats (2). Indeed, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than those of their host, and Trypanosoma brucei is no exception to this rule (3).T. brucei, the causative agent of human African trypanosomiasis (HAT), or sleeping sickness, is a blood-borne pathogenic parasite transmitted by tsetse flies. It has a complex life cycle that alternates between the bloodstream forms (BF) in the mammalian host and several stages in the insect vector starting with the procyclic form (PF) in the midgut. During T. brucei differentiation between the distinct life-cycle stages, the mitochondrion undergoes morphological and functional changes, and the parasite switches its energy metabolism from amino acid to glucose oxidation (4). BF cells, which live in sugar-rich environment, use energy metabolism predominantly through the glycolytic pathway (5). They contain no cytochrome-mediated respiratory chain and they possess a unique electron transport chain in the mitochondria, the glycerol-3-phosphate dehydrogenase and the salicyl hydroxamic acid (SHAM)-sensitive alternative oxidase, which is known as the trypanosome alternative oxidase (TAO) (6). Despite the absence of complete cytochrome-containing complexes III and IV in BF trypanosomes, a mt membrane potential is maintained and involves the hydrolytic activity of the FoF1-ATP synthase complex (7). Conversely, PF cells are dependent on the cytochrome-containing respiratory chain and ATP generated by conventional function of the FoF1-ATP synthase complex for their energy production (8, 9). The branched electron-transport chain contains four complexes that donate electrons to the ubiquinone pool, two NADH:ubiquinone oxidoreductases (complex I and a rotenone-insensitive enzyme), complex II, and glycerol-3-phosphate dehydrogenase. Reduced ubiquinol can be reoxidized by the transfer of electron to either the TAO, which does not translocate protons, or to the cytochrome-containing complexes III and IV that produce a proton motive force by translocation of protons and thus create essential membrane potential (10).Although the T. brucei genome has been sequenced (11), little information is available on the subunit composition of mt complexes I–V based on similarity searches. However, some respiratory complexes have been partially characterized in other trypanosomatids such as Crithidia fasciculata, T. cruzi, and Leishmania tarentolae (12–15). In recent studies, we have determined the protein composition of complexes IV and V, and part of complex I purified from mitochondria of T. brucei PF cells (8, 16, 17, 25). These analyses revealed the uniqueness of respiratory complexes in trypanosomes, where large numbers of component proteins have no homologs outside of the Kinetoplastida.In this study, we focus on the comprehensive characterization of all respiratory complexes in T. brucei, collectively termed the respiratome. We report the composition of complexes II and III from PF cells, and extend the characterization of complex I by identifying additional protein constituents. This included the identification of two subunits of the respiratory complex IV, both encoded by mt edited RNAs. We also present a predicted protein-protein interaction network of the respiratome, which was generated using proteomics data collected from numerous tagged proteins in each of the complexes I–V. Our results provide a comprehensive insight into the unique composition of the respiratory complexes in one of the life-cycle stages of T. brucei. 相似文献
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Chemical communication between individual Drosophila is extremely important for social behaviors required for survival and reproduction, such as con-specific recognition, courtship, aggression and avoidance of odor from "stressed" flies. Characterization of the receptors and neural circuits that detect pheromone cues and an understanding of how these circuits are modulated by the social interactions are fundamental questions about the neurobiology of social behaviors. Recent years have seen important advances in the identification of chemoreceptors and sensory neurons that are involved in sensing pheromones. Here we present a brief review of the current understanding of the peripheral chemosensory systems that are involved in social behaviors. 相似文献
65.
John Johnson Robert Okyere Anupama Joseph Karin Musier-Forsyth Besik Kankia 《Nucleic acids research》2013,41(1):220-228
Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K+ ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies. 相似文献
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Hanyoup Kim Mais J. Jebrail Anupama Sinha Zachary W. Bent Owen D. Solberg Kelly P. Williams Stanley A. Langevin Ronald F. Renzi James L. Van De Vreugde Robert J. Meagher Joseph S. Schoeniger Todd W. Lane Steven S. Branda Michael S. Bartsch Kamlesh D. Patel 《PloS one》2013,8(7)
Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories. 相似文献
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Heidi E. Godoy A. Nazmul H. Khan R. Robert Vethanayagam Melissa J. Grimm Kelly L. Singel Nonna Kolomeyevskaya Kevin J. Sexton Anupama Parameswaran Scott I. Abrams Kunle Odunsi Brahm H. Segal 《PloS one》2013,8(7)
The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function. 相似文献
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The extent of diversity among bitter-sensing neurons is a fundamental issue in the field of taste. Data are limited and conflicting as to whether bitter neurons are broadly tuned and uniform, resulting in indiscriminate avoidance of bitter stimuli, or diverse, allowing a more discerning evaluation of food sources. We provide a systematic analysis of how bitter taste is encoded by the major taste organ of the Drosophila head, the labellum. Each of 16 bitter compounds is tested physiologically against all 31 taste hairs, revealing responses that are diverse in magnitude and dynamics. Four functional classes of bitter neurons are defined. Four corresponding classes are defined through expression analysis of all 68 gustatory taste receptors. A receptor-to-neuron-to-tastant map is constructed. Misexpression of one receptor confers bitter responses as predicted by the map. These results reveal a degree of complexity that greatly expands the capacity of the system to encode bitter taste. 相似文献