Coxsackievirus B3 (CVB3) is the primary pathogen of viral myocarditis. Upon infection, CVB3 exploits the host cellular machineries, such as chaperone proteins, to benefit its own infection cycles. Inducible heat shock 70-kDa proteins (Hsp70s) are chaperone proteins induced by various cellular stress conditions. The internal ribosomal entry site (IRES) within Hsp70 mRNA allows Hsp70 to be translated cap-independently during CVB3 infection when global cap-dependent translation is compromised. The Hsp70 protein family contains two major members, Hsp70-1 and Hsp70-2. This study showed that Hsp70-1, but not Hsp70-2, was upregulated during CVB3 infection both in vitro and in vivo. Then a novel mechanism of Hsp70-1 induction was revealed in which CaMKIIγ is activated by CVB3 replication and leads to phosphorylation of heat shock factor 1 (HSF1) specifically at Serine 230, which enhances Hsp70-1 transcription. Meanwhile, phosphorylation of Ser230 induces translocation of HSF1 from the cytoplasm to nucleus, thus blocking the ERK1/2-mediated phosphorylation of HSF1 at Ser307, a negative regulatory process of Hsp70 transcription, further contributing to Hsp70-1 upregulation. Finally, we demonstrated that Hsp70-1 upregulation, in turn, stabilizes CVB3 genome via the AU-rich element (ARE) harbored in the 3' untranslated region of CVB3 genomic RNA.

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg 2؉ RNase E is a 5=-end-dependent endoribonuclease that plays a central role in stable RNA processing and mRNA turnover in Escherichia coli (1). RNase E and its paralog, RNase G, are found in many bacteria but not all (1, 2). In common with many intracellular enzymes of nucleic acid metabolism, RNase E requires a divalent metal ion for activity; in addition, it also requires Zn 2ϩ to stabilize its quaternary structure (3-5). Pioneering work by Misra and Apirion on RNase E demonstrated that the partially purified enzyme requires divalent metal ions with a preference for Mn 2ϩ over Mg 2ϩ (5). With 9S pre-RNA as the substrate, these authors reported optimal concentrations of Mn 2ϩ and Mg 2ϩ as 1 and 5 mM, respectively. Later, Redko et al. used a 3=-fluorescein-labeled decaribonucleotide (BR10F) as the substrate for the purified catalytic domain of RNase E (residues 1 to 529) and reported the optimal Mg 2ϩ concentration as 25 mM (6). Subsequently, the crystal structure of the catalytic domain of RNase E (4) revealed details of how divalent ions contribute to the activity of RNase E. Two conserved residues in the catalytic core, D303 and D346, serve to chelate a Mg 2ϩ ion, while N305 donates an H-bond that helps to anchor D303 (4). The hydration shell surrounding the bound Mg 2ϩ likely serves as the source of a hydroxyl ion that attacks the scissile phosphate (7). In addition, the bound metal ion likely polarizes the phosphate to enhance its reactivity.The ability of RNase E to utilize alternative metal ions in vivo has not been explored. The intracellular concentration of Mg 2ϩ in E. coli can reach almost 200 mM (8, 9); however, most Mg 2ϩ ions are chelated (e.g., by ribosomes [10,11]) and the free concentration is only 1 to 5 mM (8)(9)(10)12). This implies that the activity of RNase E may be limited by the availability of divalent metal ions. However, since RNA binds Mg 2ϩ , the interaction of substrates with RNase E may increase the local concentration of metal ions (7). The concentration of free Mn 2ϩ inside E. coli has not been reported to our knowledge, but total Mn 2ϩ can range from 15 M in cells cultured in unsupplemented defined medium to 150 M in rich medium thanks to active transport mechanisms (13,14). Most intracellular Mn 2ϩ is likely to be chelated resulting in a free pool whose concentration lies in the low micromolar range. Moreover, although Mn 2ϩ is a requirement for pathogenesis in related organisms such as Salmonella enterica serovar Typhimurium (13), and at least 70 enzymes are reported to be able to utilize Mn 2ϩ in E. coli (www.ecocyc.org), relatively few enzymes in E. coli absolutely require Mn 2ϩ . Such enzymes include Mn-dependent superoxide dismutase (encoded by sodA), several glycolytic enzymes and guanosine 3=-diphosphate 5=-triphosphate 3=-diphosphatase encoded by spoT (13).We...

Objective. To explore rheumatologists' perceptions of patient decision aids (PtDAs) and identify barriers to using them in clinical practice. Methods. A cross-sectional online survey of all members of the Canadian Rheumatology Association (CRA; n 5 459) was conducted. We subsequently invited 10 respondents to participate in a 30-minute telephone interview to further explore their views on using PtDAs in clinical practice. Interview participants were purposefully sampled to achieve a balance in sex, years in clinical practice, and types of practice. Results. In August and September 2013, 153 CRA members responded to the survey (response rate 33.3%); of those, 113 completed the entire questionnaire. Sixty-three respondents (55.8%) were male, 54 (47.8%) were ‡50 years of age, and 55 (48.7%) practiced in a multidisciplinary setting. When asked about their intention to use PtDAs, participants rated mean 6 SD 5.7 6 2.9 (where 0 5 not likely and 10 5 very likely). Sixty-four (56.6%) believed that rheumatologists were unfamiliar with PtDAs, and 76 (67.3%) thought that PtDAs would disturb their workflow. In-depth interviews revealed the following: the perception that PtDAs were no different from any other patient education tools, the concern that PtDAs were of limited value in real life since they relied solely on data from randomized controlled trials, and the fear that PtDAs could impair doctor-patient communication.Conclusion. There was a sense of ambivalence among rheumatologists about PtDAs. Our interviews further revealed concerns regarding the utility and benefits of PtDAs in clinical practice. The results show a need to familiarize physicians with PtDAs and to develop strategies to support their integration in clinical practice.