Integrative Functional Genomics of Hepatitis C Virus Infection Identifies Host Dependencies in Complete Viral Replication Cycle

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.     

Regeneration of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 μM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 μM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.     

Caffeine and a selective adenosine A<Subscript>2A</Subscript> receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

Background  

Caffeine, a nonselective adenosine A₁ and A_2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A_2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinson's disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A_2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum.     

Design and efficient synthesis of novel arylthiourea derivatives as potent hepatitis C virus inhibitors

A novel class of arylthiourea HCV inhibitors bearing various functionalities, such as cyclic urea, cyclic thiourea, urea, and thiourea, on the alkyl linker were designed and synthesized. Herein we report the synthesis and structure–activity relationships (SARs) of this novel class of arylthiourea derivatives that showed potent inhibitory activities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the new carbazole derivative 64, which has an eight-carbon linkage between the phenyl and carbazole rings and a tolyl group at the N-9 position of carbazole, was found to possess strong anti-HCV activity (EC₅₀ = 0.031 μM), lower cytotoxicity (CC₅₀ >50 μM), and higher selectivity index (SI >1612) compared to its derivatives.     

Membrane fouling in sterile filtration of recombinant human growth hormone

The most troublesome problem encountered during the sterile filtration of protein solutions is membrane fouling. This article presents our study on sterile filtration of a model protein, recombinant human growth hormone (rhGH). Scanning electron microscopy (SEM) analysis shows that 0.22-mum membranes, when used to filter the mannitol-formulated protein solution under a 0.35-bar transmembrane pressure, were plugged to a great extent. When zinc ions were added to induce aggregates, the fouling tendency of rhGH solutions increased with increasing amount and size of the aggregates, indicating that the aggregates present before filtration might be responsible for membrane fouling. However, repeated filtration of the same solution using a fresh filter each time cannot reduce membrane fouling, and all filtrates contain the same trace amount of hGH particulates as the prefiltered solution. Particulate size was determined to be between 0.03 and 0.15 mum by dynamic light scattering. Also, in view of the fact that protein formulations significantly affected the tendency of fouling with the same preexisting aggregates, it is likely that fouling was more attributed to the aggregation taking place in the filter pores during filtration (secondary aggregation) than to the aggregates present before filtration. Adding a surfactant to or increasing the pH of the protein solution improves the filtration, whereas increasing ionic strength slows down the filtration. This result suggests that the balance of the protein's interaction and electrostatic repulsion plays an important role in the protein's fouling tendency. Many factors might change the microenvironment in the pores and disturb this balance. Those considerations and the aggregation tendency of rhGH in the filter pores will be discussed in detail separately. (c) 1996 John Wiley & Sons, Inc.     

You stay,but I Hop: Host shifting near and far co‐dominated the evolution of Enchenopa treehoppers

The importance and prevalence of phylogenetic tracking between hosts and dependent organisms caused by co‐evolution and shifting between closely related host species have been debated for decades. Most studies of phylogenetic tracking among phytophagous insects and their host plants have been limited to insects feeding on a narrow range of host species. However, narrow host ranges can confound phylogenetic tracking (phylogenetic tracking hypothesis) with host shifting between hosts of intermediate relationship (intermediate hypothesis). Here, we investigated the evolutionary history of the Enchenopa binotata complex of treehoppers. Each species in this complex has high host fidelity, but the entire complex uses hosts across eight plant orders. The phylogenies of E. binotata were reconstructed to evaluate whether (1) tracking host phylogeny; or (2) shifting between intermediately related host plants better explains the evolutionary history of E. binotata. Our results suggest that E. binotata primarily shifted between both distant and intermediate host plants regardless of host phylogeny and less frequently tracked the phylogeny of their hosts. These findings indicate that phytophagous insects with high host fidelity, such as E. binotata, are capable of adaptation not only to closely related host plants but also to novel hosts, likely with diverse phenology and defense mechanisms.     

Uncovering the regeneration strategies of zebrafish organs: a comprehensive systems biology study on heart,cerebellum, fin,and retina regeneration

Background

Regeneration is an important biological process for the restoration of organ mass, structure, and function after damage, and involves complex bio-physiological mechanisms including cell differentiation and immune responses. We constructed four regenerative protein-protein interaction (PPI) networks using dynamic models and AIC (Akaike’s Information Criterion), based on time-course microarray data from the regeneration of four zebrafish organs: heart, cerebellum, fin, and retina. We extracted core and organ-specific proteins, and proposed a recalled-blastema-like formation model to uncover regeneration strategies in zebrafish.

Results

It was observed that the core proteins were involved in TGF-β signaling for each step in the recalled-blastema-like formation model and TGF-β signaling may be vital for regeneration. Integrins, FGF, and PDGF accelerate hemostasis during heart injury, while Bdnf shields retinal neurons from secondary damage and augments survival during the injury response. Wnt signaling mediates the growth and differentiation of cerebellum and fin neural stem cells, potentially providing a signal to trigger differentiation.

Conclusion

Through our analysis of all four zebrafish regenerative PPI networks, we provide insights that uncover the underlying strategies of zebrafish organ regeneration.

     

N-Acetyl-d-glucosamine 2-epimerase from Anabaena sp. CH1 contains a novel ATP-binding site required for catalytic activity

ATP is required as a structural activator for the reversible epimerization of N-acetyl-d-glucosamine to N-acetyl-d-mannosamine by N-acetyl-d-glucosamine 2-epimerase (AGE); however, the ATP-binding site on AGE has not been clearly identified. This study aimed to investigate the specific region of Anabaena sp. CH1 AGE (bAGE) that is required for ATP binding. In the absence of ATP, tryptic digest of bAGE resulted in the production of 2 segments of 17 and 26 kDa, while in the presence of 1 mM ATP, the enzyme was resistant to trypsin. ADP also displayed protective effects against trypsin digestion. A trypsin-mediated ATP-footprinting assay identified a deviant ATP-protected region, 156-GKYTK-160, which is located within the flexible loop of bAGE. Site-directed mutagenesis of residues in the loop region was performed, and both K151A and K160A variants greatly decreased the enzymatic activity as well as the ATP-binding ability of bAGE, indicating that residues K151 and K160 may be critical for ATP binding. This study demonstrated that the ATP-binding site (151-KDNPKGKYTK-160) of bAGE was a novel rather than a classical Walker motif A. This is the first ATP-binding site reported for AGEs.