Identification of the nature of reading frame transitions observed in prokaryotic genomes

Our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. We used the ab initio frameshift prediction program, GeneTack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. We grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position of the frameshift and its direction. We identified 4010 pseudogene clusters and 146 clusters of fs-genes apparently using recoding (local deviation from using standard genetic code) due to possessing specific sequence motifs near frameshift positions. Particularly interesting was finding of a novel type of organization of the dnaX gene, where recoding is required for synthesis of the longer subunit, tau. We selected 20 clusters of predicted recoding candidates and designed a series of genetic constructs with a reporter gene or affinity tag whose expression would require a frameshift event. Expression of the constructs in Escherichia coli demonstrated enrichment of the set of candidates with sequences that trigger genuine programmed ribosomal frameshifting; we have experimentally confirmed four new families of programmed frameshifts

Efficacy and safety of baricitinib or ravulizumab in adult patients with severe COVID-19 (TACTIC-R): a randomised, parallel-arm, open-label, phase 4 trial

Background From early in the COVID-19 pandemic, evidence suggested a role for cytokine dysregulation and complement activation in severe disease. In the TACTIC-R trial, we evaluated the efficacy and safety of baricitinib, an inhibitor of Janus kinase 1 (JAK1) and JAK2, and ravulizumab, a monoclonal inhibitor of complement C5 activation, as an adjunct to standard of care for the treatment of adult patients hospitalised with COVID-19. Methods TACTIC-R was a phase 4, randomised, parallel-arm, open-label platform trial that was undertaken in the UK with urgent public health designation to assess the potential of repurposing immunosuppressants for the treatment of severe COVID-19, stratified by a risk score. Adult participants (aged ≥18 years) were enrolled from 22 hospitals across the UK. Patients with a risk score indicating a 40% risk of admission to an intensive care unit or death were randomly assigned 1:1:1 to standard of care alone, standard of care with baricitinib, or standard of care with ravulizumab. The composite primary outcome was the time from randomisation to incidence (up to and including day 14) of the first event of death, invasive mechanical ventilation, extracorporeal membrane oxygenation, cardiovascular organ support, or renal failure. The primary interim analysis was triggered when 125 patient datasets were available up to day 14 in each study group and we included in the analysis all participants who were randomly assigned. The trial was registered on ClinicalTrials.gov (NCT04390464). Findings Between May 8, 2020, and May 7, 2021, 417 participants were recruited and randomly assigned to standard of care alone (145 patients), baricitinib (137 patients), or ravulizumab (135 patients). Only 54 (39%) of 137 patients in the baricitinib group received the maximum 14-day course, whereas 132 (98%) of 135 patients in the ravulizumab group received the intended dose. The trial was stopped after the primary interim analysis on grounds of futility. The estimated hazard ratio (HR) for reaching the composite primary endpoint was 1·11 (95% CI 0·62–1·99) for patients on baricitinib compared with standard of care alone, and 1·53 (0·88–2·67) for ravulizumab compared with standard of care alone. 45 serious adverse events (21 deaths) were reported in the standard-of-care group, 57 (24 deaths) in the baricitinib group, and 60 (18 deaths) in the ravulizumab group. Interpretation Neither baricitinib nor ravulizumab, as administered in this study, was effective in reducing disease severity in patients selected for severe COVID-19. Safety was similar between treatments and standard of care. The short period of dosing with baricitinib might explain the discrepancy between our findings and those of other trials. The therapeutic potential of targeting complement C5 activation product C5a, rather than the cleavage of C5, warrants further evaluation

Genetic decoding dynamics: from its exploitation to enhance or rescue gene expression to a search for relevance for early evolution

The genetic code and its readout were initially thought to be ‘frozen’ in the sense of being a fixed entity who’s decoding invariably involved standard rules. Recoding involves exceptions to these standard rules that are embedded in mRNA sequences. Elements in the mRNA can alter the meaning of specific codons or cause ribosomes to shift frame or with particular relevance to this thesis, result in ribosomes bypassing long untranslated regions. 30 – 50% of ribosomes successfully bypass a fifty-nucleotide insertion in phage T4 gene 60 mRNA. This coding gap begins with an in-frame stop codon and is flanked by matching GGA glycine codons. Bypassing is stimulated by a mixture of signals including a nascent peptide and a mRNA stem loop which includes the take-off codon at the start of the coding gap. An additional stem loop whose 3’ end is nine nucleotides 5’ of the take-off codon, was recently demonstrated to be important for bypassing in vitro. I investigated the effect of this structure in vivo. The structure is important in vivo when the number of ribosomes translating gene 60 is very small. There is good evidence that the 5’ stem loop functions to provide forward momentum for bypassing ribosomes in the 5’ region of the coding gap, and it may also possibly have a role in preventing backward movement. No effect for the 5’ stem loop was observed in high expression systems and this was deduced to be due to the following ribosome performing what is normally carried out by this structure, giving bypassing ribosomes forward momentum. Prior work claimed that mRNA structure 3’ of the site of coding resumption influenced bypassing efficiency. In vivo data reported here does not support this under the conditions tested with either multiple or few ribosomes translating the gene 60 sequence. While half to one third of ribosomes successfully bypass the non-coding sequence, earlier experiments provided strong evidence that the efficiency of peptidyl-tRNA dissociation from the take-off codon at the start of the coding gap is 100%. Therefore, the ability of ribosomes to recognise the glycine landing codon and resume translation is key for efficient bypassing. The identity of the matching codons is important for efficient bypassing with combinations of matched codons other than GGA permitting bypassing. Variants at the resume codon position had not previously been tested. The present work shows that slow-to-decode codons had the largest effect on bypassing when present as the resume codon. Bypassing and frameshifting ribosomes enter a non-canonical state during the respective recoding events, potentially affecting their ability to resume translation immediately afterwards. When bypassing ribosomes resume translation, we showed that they decode several codons before fully regaining standard translational properties, consistent with earlier more indirect inferences. Productive translational bypassing was first identified twenty-eight years ago, yet new cases of bypassing have not been abundant. Recently, 81 potential cases of bypassing were discovered in the yeast Magnusiomyces capitatus with 3 cases being experimentally validated. We have identified new bypassing candidates in Streptomyces coelicolor. These bacteria are proposed to take advantage of the presence of a rare UUA codon to stimulate bypassing. Others have provided suggestive evidence that certain Streptomyces phages also utilize translational bypassing. Extensive and costly searches for life on Mars are underway. However, no such search for radical new life has been undertaken here on Earth. The likely theory of the RNA World postulates that an RNA-organism predated the emergence of DNA and protein catalysts. We undertook a multidisciplinary search of deep earth cores and pockets of ancient water that have been isolated from modern genetic organisms. We initiated a search for niches that might contain RNA in the absence of ribosomal RNA, and much more extensive further investigations are merited

Breaking Patterns of Conflict in Northern Ireland: New Perspectives

Central to reaching peace and settlement in Northern Ireland was a sequence of British--Irish intergovernmental discussions and negotiations, dating from the beginning of the 1980s. British and Irish state cooperation and intervention has remained central to the stability of the settlement reached in 1998. The motives of state actors, however, have been unclear, and the role of the state in the political process has been the subject of some scholarly controversy. This paper looks at the types of evidence that can help to resolve such questions. It focuses on the value of elite interviews, arguing that they can constitute an important and irreplaceable body of evidence when used critically, but it also highlights the risks of excessive reliance on this type of source. It goes on to describe a major research project in University College Dublin whose aim was to record the experiences and interpretations of the actors who engaged in British–Irish negotiations over the last four decades. It discusses the resulting elite interviews and witness seminars and the methodological and ethical difficulties encountered. It describes how these were overcome, and outlines the conditions of confidentiality imposed.Author has checked copyrigh