Yeast ribosome biogenesis factors Puf6 and Nog2 and ribosomal proteins uL2 and eL43 act in concert to facilitate the release of nascent large ribosomal subunits from the nucleolus.

Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the nucleoplasm. Structural models of yeast pre-LSUs at various stages of assembly have been collected using cryo-EM. However, which cryo-EM model is closest to the final nucleolar intermediate of the LSU has yet to be determined. To elucidate the mechanisms of the release of pre-LSUs from the nucleolus, we assayed effects of depleting or knocking out two yeast ribosome biogenesis factors (RiBi factors), Puf6 and Nog2, and two ribosomal proteins, uL2 and eL43. These proteins function during or stabilize onto pre-LSUs between the late nucleolar stages to early nucleoplasmic stages of ribosome biogenesis. By characterizing the phenotype of these four mutants, we determined that a particle that is intermediate between the cryo-EM model State NE1 and State NE2 likely represents the final nucleolar assembly intermediate of the LSU. We conclude that the release of the RiBi factors Nip7, Nop2 and Spb1 and the subsequent stabilization of rRNA domains IV and V may be key triggers for the release of pre-LSUs from the nucleolus.

Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Waiting for infected hip revision surgery; exploring patient and families' experience of waiting (PREWs).

INTRODUCTION: As a tertiary center for complex orthopedic surgery, evaluating current practice is vital to enhance pathways of care. A vital element of this is to understand service users' perspectives and experiences, yet little research to date has explored experiences of waiting for surgery in this cohort. METHODS: The aim of this mixed methods study was to understand patients' and families' experiences of waiting for hip revision surgery for joint infection and their perceptions of its impact on their quality of life. Data were collected via a survey and individual interviews with people who had experienced waiting for surgery between May 01, 2018 and April 30, 2019. Of the 54 participants invited to participate, 22 returned a questionnaire and five were interviewed. Quantitative data were analysed descriptively whilst qualitative data underwent thematic analysis. RESULTS: Although 67 % of respondents reported being satisfied with their waiting time, all reported an increase in pain whilst waiting and 60 % stated that waiting had a significant effect on their lives and those of their friends/family (85 %). Most respondents reported high levels of satisfaction with the support (89 %) and information (83 %) provided by the clinical team. CONCLUSION: Despite satisfaction with support and information being rated highly, waiting for surgery clearly has a great impact on patients and their families. A named clinician to monitor/oversee care whilst waiting and to act as contact to help them navigate services is recommended to support people during the waiting period.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae.

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels.

Oral versus Intravenous Antibiotics for Bone and Joint Infection.

BACKGROUND: The management of complex orthopedic infections usually includes a prolonged course of intravenous antibiotic agents. We investigated whether oral antibiotic therapy is noninferior to intravenous antibiotic therapy for this indication. METHODS: We enrolled adults who were being treated for bone or joint infection at 26 U.K. centers. Within 7 days after surgery (or, if the infection was being managed without surgery, within 7 days after the start of antibiotic treatment), participants were randomly assigned to receive either intravenous or oral antibiotics to complete the first 6 weeks of therapy. Follow-on oral antibiotics were permitted in both groups. The primary end point was definitive treatment failure within 1 year after randomization. In the analysis of the risk of the primary end point, the noninferiority margin was 7.5 percentage points. RESULTS: Among the 1054 participants (527 in each group), end-point data were available for 1015 (96.3%). Treatment failure occurred in 74 of 506 participants (14.6%) in the intravenous group and 67 of 509 participants (13.2%) in the oral group. Missing end-point data (39 participants, 3.7%) were imputed. The intention-to-treat analysis showed a difference in the risk of definitive treatment failure (oral group vs. intravenous group) of -1.4 percentage points (90% confidence interval [CI], -4.9 to 2.2; 95% CI, -5.6 to 2.9), indicating noninferiority. Complete-case, per-protocol, and sensitivity analyses supported this result. The between-group difference in the incidence of serious adverse events was not significant (146 of 527 participants [27.7%] in the intravenous group and 138 of 527 [26.2%] in the oral group; P=0.58). Catheter complications, analyzed as a secondary end point, were more common in the intravenous group (9.4% vs. 1.0%). CONCLUSIONS: Oral antibiotic therapy was noninferior to intravenous antibiotic therapy when used during the first 6 weeks for complex orthopedic infection, as assessed by treatment failure at 1 year. (Funded by the National Institute for Health Research; OVIVA Current Controlled Trials number, ISRCTN91566927 .).

Successful implementation of an automated electronic support system for patient safety monitoring: The alemtuzumab in multiple sclerosis safety systems (AMS3) study.

BACKGROUND: Alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis (MS) but requires ongoing pathology monitoring for autoimmune adverse effects. The Alemtuzumab in MS Safety Systems (AMS3) study evaluated the implementation of an automated pathology-monitoring system. OBJECTIVES: To develop an efficient automated clinical decision support system (CDSS) to electronically prompt and track pathology collection and to provide prescribers and patients with customised alerts of abnormal results for identified risks. METHODS: A total of 10 patients with relapsing-remitting MS treated with alemtuzumab were enrolled to test the system. Standard care laboratory monitoring was performed and compared to the performance of the CDSS. RESULTS: The automated CDSS, an integrated patient smartphone application and an additional pre-screening tool were all successfully developed. Compliance with pathology monitoring was 96.7%. The automated analysis of pathology results was significantly faster than standard care neurologist review (p < 0.001). The system correctly identified and alerted abnormalities, including one case of immune thrombocytopenia (ITP) while the treating neurologist was on leave, enabling prompt treatment of serious adverse events. During the course of the study, the CDSS was deployed throughout Australia. CONCLUSION: We successfully developed automated pathology monitoring with a CDSS, demonstrating real-world benefits of high compliance and timely alerting of important results.