Investigating a New Generation of Ribozymes in Order to Target HCV

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity “on” solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (−) strands

A molecular insight into algal-oomycete warfare : cDNA analysis of Ectocarpus siliculosus infected with the basal oomycete Eurychasma dicksonii

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Can bryophyte groups increase functional resolution in tundra ecosystems?

Funding Information: This study was supported by a grant to SL from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie, Grant No. 797446 and by the Independent Research Fund Denmark, Grant no. 0135-00140B. Funding from the Academy of Finland (grant 322266), National Science Foundation (1504224, 1836839, PLR-1504381 and PLR-1836898), Independent Research Fund Denmark (9040-00314B), Moscow State University, (project No 121032500089-1), Natural Sciences and Engineering Research Council of Canada, ArcticNet, Polar Continental Shelf Program, Northern Science Training Program, Polar Knowledge Canada, Royal Canadian Mounted Police, Tomsk State University competitiveness improvement program and the Russian Science Foundation (grant No 20-67-46018) are gratefully acknowledged. Matthias Ahrens provided valuable insights on the cushion growth form, and we are most thankful. We thank Gaius Shaver and two anonymous reviewers for providing valuable critique and input to earlier versions of this manuscript. Publisher Copyright: © the author(s) or their institution(s).The relative contribution of bryophytes to plant diversity, primary productivity, and ecosystem functioning increases towards colder climates. Bryophytes respond to environmental changes at the species level, but because bryophyte species are relatively difficult to identify, they are often lumped into one functional group. Consequently, bryophyte function remains poorly resolved. Here, we explore how higher resolution of bryophyte functional diversity can be encouraged and implemented in tundra ecological studies. We briefly review previous bryophyte functional classifications and the roles of bryophytes in tundra ecosystems and their susceptibility to environmental change. Based on shoot morphology and colony organization, we then propose twelve easily distinguishable bryophyte functional groups. To illustrate how bryophyte functional groups can help elucidate variation in bryophyte effects and responses, we compiled existing data on water holding capacity, a key bryophyte trait. Although plant functional groups can mask potentially high interspecific and intraspecific variability, we found better separation of bryophyte functional group means compared with previous grouping systems regarding water holding capacity. This suggests that our bryophyte functional groups truly represent variation in the functional roles of bryophytes in tundra ecosystems. Lastly, we provide recommendations to improve the monitoring of bryophyte community changes in tundra study sites.Peer reviewe

Gambling Problems among Community Cocaine Users

Cocaine use is highly prevalent and a major public health problem. While some studies have reported frequent comorbidity problems among cocaine users, few studies have included evaluation of gambling problems. This study aimed to estimate the prevalence of gambling problems and compare those who were at-risk gamblers with non-problem gamblers in terms of mental health problems, substance use problems, and some risk factors (i.e. family antecedents, erroneous perceptions and coping strategies) among individuals who smoke or inject cocaine. METHOD: A total of 424 smoked or injected cocaine users recruited through community-based programs in Montreal, Quebec completed the questionnaire, including the Canadian Pathological Gambling Index, the Composite International Diagnostic Interview (CIDI), the CAGE, and the Severity Dependence Scale (SDS). RESULTS: Of the sample, 18.4 % were considered at-risk gamblers, of whom 7.8 % had problems gambling and 10.6 % were moderate-risk gamblers. The at-risk group was more likely to have experienced a recent phobic disorder and alcohol problems than the non-problem group. A multivariate analysis showed that, compared to those who were non-problem gamblers, the at-risk ones were more likely to have lost a large sum of money when they first started gambling, believed that their luck would turn, and gambled in reaction to painful life events. These results indicate the need to include routines for screening to identify gambling problem among cocaine user

Can bryophyte groups increase functional resolution in tundra ecosystems?

The relative contribution of bryophytes to plant diversity, primary productivity, and ecosystem functioning increases towards colder climates. Bryophytes respond to environmental changes at the species level, but because bryophyte species are relatively difficult to identify, they are often lumped into one functional group. Consequently, bryophyte function remains poorly resolved. Here, we explore how higher resolution of bryophyte functional diversity can be encouraged and implemented in tundra ecological studies. We briefly review previous bryophyte functional classifications and the roles of bryophytes in tundra ecosystems and their susceptibility to environmental change. Based on shoot morphology and colony organization, we then propose twelve easily distinguishable bryophyte functional groups. To illustrate how bryophyte functional groups can help elucidate variation in bryophyte effects and responses, we compiled existing data on water holding capacity, a key bryophyte trait. Although plant functional groups can mask potentially high interspecific and intraspecific variability, we found better separation of bryophyte functional group means compared with previous grouping systems regarding water holding capacity. This suggests that our bryophyte functional groups truly represent variation in the functional roles of bryophytes in tundra ecosystems. Lastly, we provide recommendations to improve the monitoring of bryophyte community changes in tundra study sites

Schematic representation of the 3-step procedure used for the identification of the sites possessing the greatest targeting potential in the HCV 5′-UTR.

<p>Step 1 involved a bioinformatic analysis that included both the prediction of the secondary structure and the identification of the 7 nt streches most likely to be bound by the ribozyme's P1 region using both the RNA structure 3.7 and Oligowalk softwares. Step 2 involved the selection of the sequences that fulfill the HDV ribozyme requirements. Step 3 involved the RNase H hydrolysis assay. The autoradiogram shown corresponds to a typical 5% polyacrylamide gel performed for the analysis of 6 potential sites. The positions of proposed cleavage sites are identified at the top of the gel. The negative control performed in the absence of any oligonucleotide is indicated by the letter C.</p

Compilation of the in vitro cleavage of HCV (+) strand by the SOFA-HDV ribozyme data.

<p>The sequences for each SOFA-HDV Rz corresponding to their P1 and biosensor recognition domains are listed. The upper section includes the SOFA-HDV Rz targetable sites as identified by bioinformatic and biochemical procedures, while the lower section includes those based on the reported secondary and crystal structures.</p

Analysis of the inhibition of the HCV replicon by SOFA-HDV ribozymes.

<p>(A) Schematic representation of the HCV replicon used (described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009627#pone.0009627-Vrolijk1" target="_blank">[38]</a>). (B) and (C). Histograms of the relative luciferase activities detected for the HCV (+) and (−) strands, respectively. Luciferase activity was detected in the presence of all SOFA-HDV ribozymes tested, and was reported relative to that determined for an irrelevant SOFA-HDV ribozyme whose level was arbitrarily set at 100%. The latter SOFA-HDV ribozyme was designed to target the hepatitis B virus and does not possess the sequences required in order to recognize the HCV strands of either the (+) or the (−) polarity. The characterization of this SOFA-HDV ribozyme was previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009627#pone.0009627-Bergeron1" target="_blank">[29]</a>.</p

Typical autoradiogram of an 8% polyacrylamide gel performed in order to analyze SOFA-HDV ribozyme cleavage <i>in vitro</i>.

<p>The experiments were performed using 5′-end-labeled HCV transcripts in the presence of an excess of SOFA-HDV ribozyme. The SOFA-HDV ribozymes are identified at the top of the gel. The negative control performed in the absence of ribozyme is indicated by the letter C. The positions of the xylene cyanol (XC) and bromophenol blue (BPB) marker dyes are indicated.</p

Compilation of the data from the RNase H hydrolysis of HCV (+) RNA.

<p>Compilation of the data from the RNase H hydrolysis of HCV (+) RNA.</p