RNA-seq transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis

Bovine tuberculosis, caused by infection with Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including gene expression microarrays and RNA-sequencing (RNA-seq), has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analyzed the peripheral blood leukocyte (PBL) transcriptome of eight natural M. bovis-infected and eight age- and sex-matched non-infected control Holstein-Friesian animals using RNA-seq. In addition, we compared gene expression profiles generated using RNA-seq with those previously generated using the high-density Affymetrix(®) GeneChip(®) Bovine Genome Array platform from the same PBL-extracted RNA. A total of 3,250 differentially expressed (DE) annotated genes were detected in the M. bovis-infected samples relative to the controls (adjusted P-value ≤0.05), with the number of genes displaying decreased relative expression (1,671) exceeding those with increased relative expression (1,579). Ingenuity(®) Systems Pathway Analysis (IPA) of all DE genes revealed enrichment for genes with immune function. Notably, transcriptional suppression was observed among several of the top-ranking canonical pathways including Leukocyte Extravasation Signaling. Comparative platform analysis demonstrated that RNA-seq detected a larger number of annotated DE genes (3,250) relative to the microarray (1,398), of which 917 genes were common to both technologies and displayed the same direction of expression. Finally, we show that RNA-seq had an increased dynamic range compared to the microarray for estimating differential gene expression

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA

New graduate doctors' preparedness for practice: A multistakeholder, multicentre narrative study

This is the final version. Available on open access from BMJ Publishing Group via the link in this recordData sharing statement The raw data for this research consist of audio-recordings of narrative interviews and audio diaries. The principal investigator (Professor Lynn V Monrouxe) has access to this specific data set, including audio-recordings of interviews and interview transcripts, in addition to participant contact details and signed consent forms. All authors have access to anonymised data from this set. All data are stored securely on password-protected and encrypted computers. Participants have not given their permission for data sharing outside the research group. Thus, no additional data are available.Objective While previous studies have begun to explore newly graduated junior doctors' preparedness for practice, findings are largely based on simplistic survey data or perceptions of newly graduated junior doctors and their clinical supervisors alone. This study explores, in a deeper manner, multiple stakeholders' conceptualisations of what it means to be prepared for practice and their perceptions about newly graduated junior doctors' preparedness (or unpreparedness) using innovative qualitative methods. Design A multistakeholder, multicentre qualitative study including narrative interviews and longitudinal audio diaries. Setting Four UK settings: England, Northern Ireland, Scotland and Wales. Participants Eight stakeholder groups comprising n=185 participants engaged in 101 narrative interviews (27 group and 84 individual). Twenty-six junior doctors in their first year postgraduation also provided audio diaries over a 3-month period. Results We identified 2186 narratives across all participants (506 classified as 'prepared', 663 as 'unprepared', 951 as 'general'). Seven themes were identified; this paper focuses on two themes pertinent to our research questions: (1) explicit conceptualisations of preparedness for practice; and (2) newly graduated junior doctors' preparedness for the General Medical Council's (GMC) outcomes for graduates. Stakeholders' conceptualisations of preparedness for practice included short-term (hitting the ground running) and long-term preparedness, alongside being prepared for practical and emotional aspects. Stakeholders' perceptions of medical graduates' preparedness for practice varied across different GMC outcomes for graduates (eg, Doctor as Scholar and Scientist, as Practitioner, as Professional) and across stakeholders (eg, newly graduated doctors sometimes perceived themselves as prepared but others did not). Conclusion Our narrative findings highlight the complexities and nuances surrounding new medical graduates' preparedness for practice. We encourage stakeholders to develop a shared understanding (and realistic expectations) of new medical graduates' preparedness. We invite medical school leaders to increase the proportion of time that medical students spend participating meaningfully in multiprofessional teams during workplace learning.General Medical Counci

Decentralised Collective Bargaining in Ireland - National Report

The institutional framework for collective bargaining in Ireland is underpinned by the principle of voluntarism. Employers and trade unions voluntarily engage in collective bargaining, and their agreed terms and conditions of employment are not legally binding. Workers have the right to form and join a trade union. However, unions cannot force employers to enter collective bargaining, meaning that there is no legal right to bargaining in Ireland. There is only one trade union confederation, the Irish Congress of Trade Unions. Equally, there is one major cross-sectoral employers’ association, the Irish Business and Employers Confederation. These two organisations played a very significant role in the regulation of the economy during the national social partnership in Ireland between 1987 and 2009, negotiating wage rates for all unionised workers. Since the collapse of the national social partnership in 2009, the main levels at which collective bargaining takes place are the company and the workplace levels. Sectoral bargaining still occurs in a number of low-paid and weakly unionised sectors and in construction and allied sectors. Sectoral bargaining also takes place in public services. Recently, employers and unions have called for a deepening of tripartite social dialogue, and this has occurred in several areas central to Ireland’s response to the COVID-19 pandemic. However, this emerging forum cannot be considered as a precursor to a new form of national social partnership. Neither unions nor employers favour the alignment of social dialogue directly with pay bargaining in a renewal of the social partnership-type centralised pay agreements of the 1987–2009 era. The main actors involved in decentralised bargaining are, on the side of the union, full-time trade union officials, organising workers at the sector level alongside shop stewards working in companies, and, on the side of the employer, Human Resource (HR) Directors and their delegation of managers, variously composed of the HR Manager(s), Head of Finance, and Operations Manager(s). In the largest companies, which are considered to be pattern setters in collective bargaining, the employers’ association also takes part in the negotiations. There are no works councils within companies in Ireland, and only trade unions with a licence are authorised to sign a collective agreement with their management counterpart. In some companies, particular industrial relations arrangements, whereby trade unions and management collaborate in various areas, such as information sharing, training, and work organisation, can be found. These are called workplace partnerships and are supported by formal workplace participation forums that are used by both management and shop stewards to voice their concerns as well as to discuss any issue that is relevant to the workers and the company. These forums, however, are never the locus of collective negotiations

Expanding the role of participatory mapping to assess ecosystem service provision in local coastal environments

There has been increasing international effort to better understand the diversity and quality of marine natural capital, ecosystem services and their associated societal benefits. However, there is an evidence gap as to how these benefits are identified at the local scale, where benefits are provided and to whom, trade-offs in development decisions, and understanding how benefits support well-being. Often the benefits of conservation are poorly understood at the local scale, are not effectively integrated into policy and are rarely included meaningfully in public discourse. This paper addresses this disjuncture and responds to the demand for improving dialogue with local communities and stakeholders. Participatory GIS mapping is used as a direct means of co-producing knowledge with stakeholder and community interests. This paper drives a shift from development of participatory approaches to adaptive applications in real-world case studies of local, national and international policy relevance. The results from four sites along the UK North Sea coast are presented. This paper showcases a robust stakeholder-driven approach that can be used to inform marine planning, conservation management and coastal development. Although the demonstration sites are UK-focused, the methodology presented is of global significance and can be applied across spatial and temporal scales

How do pilot and feasibility studies inform randomised placebo-controlled trials in surgery? : A systematic review

© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.Peer reviewedPublisher PD