Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC₅₀s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99⁸²A, I59⁵⁰V, and V37³²I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC₅₀ of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.     

Meta-analysis of Gene-Level Associations for Rare Variants Based on Single-Variant Statistics

Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying “causal” rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available.     

Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity

Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2′-phosphate of NADPH, but also could enhance the binding with 2′-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2′-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity.     

O-Heterocyclic Embeurekols from Embellisia eureka,an Endophyte of Cladanthus arabicus

Three new polyketides (-)-1 , (+)-1 , and 2 ) were isolated from the EtOAc extract of the fungus Embellisia eureka, an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of (-)-1 , (+)-1 , and 2 were determined by TDDFT ECD calculations of solution conformers, online HPLC-ECD analysis, and the modified Mosher method. Chirality 25:250–256, 2013. © 2013 Wiley Periodicals, Inc.     

Design and synthesis of 1-aryl-5-anilinoindazoles as c-Jun N-terminal kinase inhibitors

Starting from pyrazole HTS hit (1), a series of 1-aryl-1H-indazoles have been synthesized as JNK3 inhibitors with moderate selectivity against JNK1. SAR studies led to the synthesis of 5r as double digital nanomolar JNK3 inhibitor with good in vivo exposure.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Morphology of three new marine Frontonia species (Ciliophora; Peniculida) with note on the phylogeny of this genus

Members of the ciliate genus Frontonia are common colonizers of periphytic communities in aquatic biotopes. Recent studies indicate that their species diversity is higher than previously supposed. In this study the morphology and infraciliature of three new species, Frontonia sinica spec. nov., F. pusilla spec. nov., and F. elegans spec. nov., isolated from coastal waters of China, were investigated using live observation and silver impregnation methods. Frontonia sinica differs from its congeners by the following combination of characters: ellipsoidal body, about 116 somatic and five or six vestibular kineties, peniculi 1 and 2 four-rowed, peniculus 3 two-rowed, and a single contractile vacuole. Frontonia pusilla has about 72 somatic kineties, four-rowed peniculi 1 and 2, a two-rowed peniculus 3, and two contractile vacuoles. Frontonia elegans has 73 somatic kineties, four-rowed peniculi 1 and 2, a three-rowed peniculus 3, and two contractile vacuoles. In the present work, six new small-subunit rRNA gene sequences of six Frontonia species are used to construct the phylogenetic trees. Our phylogenetic analysis supports that the genus Frontonia may be paraphyletic. Meanwhile, no pattern of correlation could be found between the structures of peniculi and the phylogenetic relationships of Frontonia species in the present study.