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

Louise Alphonsine Nantel, journaliste au tournant du XXe siècle

La journaliste Louise Alphonsine Nantel (1884-1965) a rédigé de nombreuses chroniques dans L’Avenir du Nord, Le Canada, Le Pays et La Patrie signées Andrée Claudel, Mireille, Lou Sorriaux ou L. A. Nantel. Le présent article se penche sur ses premiers écrits, de 1904 à 1914. Née à Saint-Jérôme, Louise Alphonsine déménage à Montréal à 19 ans puis s’installe à Paris en 1911, d’où elle poursuit sa collaboration aux journaux montréalais. On lui doit des portraits saisissants de la vie urbaine, et, si elle publie bon nombre de chroniques qu’on peut qualifier de mondaines, elle demeure critique envers la frivolité et l’hypocrisie de la bourgeoisie. Femme moderne et émancipée, elle appartient au mouvement progressiste; elle soutient les revendications féministes ainsi que la participation des femmes à la vie publique et plaide pour une bibliothèque publique et l’appui du gouvernement à l’éducation. Elle épouse le commissaire de police parisien Julien Sorriaux. En 1930, le couple s’établit à Saint-Tropez, où la journaliste meurt en 1966. En 1947, elle publie un roman autobiographique sur son enfance dans les Laurentides.Journalist Louise Alphonsine Nantel (1884–1965) published many columns in L’Avenir du Nord, Le Canada, Le Pays and La Patrie under the names of Andrée Claudel, Mireille, Lou Sorriaux and L.A. Nantel. This article examines her early work (1904–1914). Born in Saint-Jérôme, Louise Alphonsine moved to Montreal at the age of 19 and then settled in Paris in 1911, from where she continued collaborating with Montreal newspapers. She is the author of vivid portraits of urban life, and while she published a large number of “social columns,” she remained critical of the bourgeoisie’s frivolity and hypocrisy. A modern and emancipated woman, she belonged to the progressive movement: she supported feminist demands as well as the participation of women in public life, and advocated for a public library and the government’s support of education. She married a Parisian chief of police Julien Sorriaux. In 1930, the couple moved to Saint-Tropez where the journalist passed away in 1966. In 1947, she published an autobiographical novel about her childhood in the Laurentides.La journaliste Louise Alphonsine Nantel (1884-1965) a rédigé de nombreuses chroniques dans L’Avenir du Nord, Le Canada, Le Pays et La Patrie signées Andrée Claudel, Mireille, Lou Sorriaux ou L. A. Nantel. Le présent article se penche sur ses premiers écrits, de 1904 à 1914. Née à Saint-Jérôme, Louise Alphonsine déménage à Montréal à 19 ans puis s’installe à Paris en 1911, d’où elle poursuit sa collaboration aux journaux montréalais. On lui doit des portraits saisissants de la vie urbaine, et, si elle publie bon nombre de chroniques qu’on peut qualifier de mondaines, elle demeure critique envers la frivolité et l’hypocrisie de la bourgeoisie. Femme moderne et émancipée, elle appartient au mouvement progressiste; elle soutient les revendications féministes ainsi que la participation des femmes à la vie publique et plaide pour une bibliothèque publique et l’appui du gouvernement à l’éducation. Elle épouse le commissaire de police parisien Julien Sorriaux. En 1930, le couple s’établit à Saint-Tropez, où la journaliste meurt en 1966. En 1947, elle publie un roman autobiographique sur son enfance dans les Laurentides.Journalist Louise Alphonsine Nantel (1884–1965) published many columns in L’Avenir du Nord, Le Canada, Le Pays and La Patrie under the names of Andrée Claudel, Mireille, Lou Sorriaux and L.A. Nantel. This article examines her early work (1904–1914). Born in Saint-Jérôme, Louise Alphonsine moved to Montreal at the age of 19 and then settled in Paris in 1911, from where she continued collaborating with Montreal newspapers. She is the author of vivid portraits of urban life, and while she published a large number of “social columns,” she remained critical of the bourgeoisie’s frivolity and hypocrisy. A modern and emancipated woman, she belonged to the progressive movement: she supported feminist demands as well as the participation of women in public life, and advocated for a public library and the government’s support of education. She married a Parisian chief of police Julien Sorriaux. In 1930, the couple moved to Saint-Tropez where the journalist passed away in 1966. In 1947, she published an autobiographical novel about her childhood in the Laurentides

A Modern Mode of Activation for Nucleic Acid Enzymes

Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process

Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme

The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction

Distinct Roles of Bcl-2 and Bcl-Xl in the Apoptosis of Human Bone Marrow Mesenchymal Stem Cells during Differentiation

Background: Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells. Principal Findings: Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs. Significance: In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions

The prevention and management of chronic disease in primary care: recommendations from a knowledge translation meeting

BACKGROUND: Seven chronic disease prevention and management programs were implemented across Quebec with funding support from a provincial-private industry funding initiative. Given the complexity of implementing integrated primary care chronic disease management programs, a knowledge transfer meeting was held to share experiences across programs and synthesize common challenges and success factors for implementation. METHODS: The knowledge translation meeting was held in February 2014 in Montreal, Canada. Seventy-five participants consisting of 15 clinicians, 14 researchers, 31 knowledge users, and 15 representatives from the funding agencies were broken up into groups of 10 or 11 and conducted a strengths, weaknesses, opportunities, and threats analysis on either the implementation or the evaluation of these chronic disease management programs. Results were reported back to the larger group during a plenary and recorded. Audiotapes were transcribed and summarized using pragmatic thematic analysis. RESULTS AND DISCUSSION: Strengths to leverage for the implementation of the seven programs include: (1) synergy between clinical and research teams; (2) stakeholders working together; (3) motivation of clinicians; and (4) the fact that the programs are evidence-based. Weaknesses to address include: (1) insufficient resources; (2) organizational change within the clinical sites; (3) lack of referrals from primary care physicians; and (4) lack of access to programs. Strengths to leverage for the evaluation of these programs include: (1) engagement of stakeholders and (2) sharing of knowledge between clinical sites. Weaknesses to address include: (1) lack of referrals; (2) difficulties with data collection; and (3) difficulties in identifying indicators and control groups. Opportunities for both themes include: (1) fostering new and existing partnerships and stakeholder relations; (2) seizing funding opportunities; (3) knowledge transfer; (4) supporting the transformation of professional roles; (5) expand the use of health information technology; and (6) conduct cost evaluations. Fifteen recommendations related to mobilisation of primary care physicians, support for the transformation of professional roles, and strategies aimed at facilitating the implementation and evaluation of chronic disease management programs were formulated based on the discussions at this knowledge translation event. CONCLUSION: The results from this knowledge translation day will help inform the sustainability of these seven chronic disease management programs in Quebec and the implementation and evaluation of similar programs elsewhere