Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups.

BACKGROUND: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. RESULTS: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. CONCLUSIONS: The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.

Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.

While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.

Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing.

Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals.

Managing change in postgraduate medical education: still unfreezing?

BACKGROUND: Modernizing Medical Careers (MMC) is an ambitious project to change the training of UK doctors. A key to its successful implementation is the ways that MMC is perceived and operationalized by senior doctors who act as local educational leaders and supervisors. AIMS: To analyse hospital consultants' perceptions of the modernization process and its impact on their role as the primary educators of Senior House Officers (SHOs), using Schein's extended model to explain their stage in the process of change. METHODS: We interviewed medical directors, College and clinical tutors and education supervisors at 6 Trusts. The transcripts were analysed using Schein's change model to explore the perceptions and assumptions of senior medical staff and to determine their stage in the process of change. RESULTS: 12 tutors, 12 supervisors, and 4/6 medical directors approached agreed to participate (28/30). Nine themes emerged from transcript analysis. These were related to the three-stage model of change. Most participants were at the stage of 'unfreezing', expressing views around disconfirmation of expectations, guilt and anxiety and feelings of some psychological safety. A smaller number were at the stage of 'moving to a new position'. There were limited examples of 'refreezing'. CONCLUSIONS: At the local delivery level, most senior doctors were aware of the need to review their current position and alter their approaches and assumptions about postgraduate medical education. Yet only a minority were moving forward. Considerable work remains for successful implementation of MMC.

Learning the house officer role: reflections on the value of shadowing a PRHO.

In the new integrated undergraduate medical programme at the University of Manchester, fifth-year students spend several weeks shadowing the pre-registration house officer (PRHO) whose post they will take over. The concept of 'shadowing' emerged from a set of interviews conducted with graduates during their first PRHO job. Graduates felt that shadowing helped them to gain familiarity with the work environment; with orientation to the role of a PRHO; and with specific learning, such as disease management, on which they could then get feedback. We hypothesize that shadowing provides an opportunity for focused apprenticeship learning of the future PRHO role. Further research may clarify the specific values of shadowing and how it might lessen the stresses faced by new graduates during the transition from student to doctor.

A model of how students link problem-based learning with clinical experience through "elaboration".

PURPOSE: In 1994, the medical school at the University of Manchester introduced a new integrated course that uses problem-based learning (PBL) throughout the clinical clerkships as the major approach for delivery of the core curriculum. This study explored how students linked PBL and clinical experiences. METHOD: All third- and fourth-year students were asked to respond to an open-ended question on the end-of-module course evaluation. Their responses were analyzed and grouped into themes. The authors also conducted three focus groups of third-year students and a further three focus groups of fourth-year students to explore how students viewed the links between PBL and clinical experiences. The discussions were taped and subsequently analyzed by the researchers independently. RESULTS: The authors found that the students used clinical experience as a means of elaborating their knowledge either at the time of encountering an appropriate patient (outside the group) or by bringing their experiences back to the PBL group for discussion (inside the group). Major facilitators of elaboration were the match between the clinical clerkship and the content of the PBL case, the role of the tutor, and the self-directedness of the student. A theoretical model of how students linked PBL with their clinical experience was derived based on a cognitive psychological approach to learning. CONCLUSIONS: The model will be of benefit as the authors try to improve the course for those students who were unable to use their clinical experiences to achieve the goals of their PBL discussions.

Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland.

Phytophthora lateralis is a fungus-like (oomycete) pathogen of trees in the family Cupressaceae, including Chamaecyparis lawsoniana (Lawson cypress or Port Orford cedar). Known in North America since the 1920s, presumably having been accidentally introduced from its assumed East Asian centre of origin, until recently, this pathogen has not been identified causing disease in Europe except for a few isolated outbreaks. However, since 2010, there have been several reports of infection of C. lawsoniana by P. lateralis in the United Kingdom, including Northern Ireland. We sequenced the genomes of four isolates of P. lateralis from two sites in Northern Ireland in 2011. Comparison with the closely related tree and shrub pathogen P. ramorum (cause of ramorum disease of larch and other species in the UK) shows that P. lateralis shares 91.47% nucleotide sequence identity over the core conserved compartments of the genome. The genomes of the four Northern Ireland isolates are almost identical, but we identified several single-nucleotide polymorphisms (SNPs) that distinguish between isolates, thereby presenting potential molecular markers of use for tracking routes of spread and in epidemiological studies. Our data reveal very low rates of heterozygosity (compared with P. ramorum), consistent with inbreeding within this P. lateralis population.

Views, behaviours and perceived staff development needs of doctors and surgeons regarding learners in outpatient clinics.

INTRODUCTION: The need to use outpatient clinics as a major learning environment in hospitals for students and doctors-in-training is clear. However, consultant supervisors perceive major barriers to this and continue to rely heavily on traditional inpatient learning. This quantitative study examines what approaches consultant supervisors employ in outpatient learning, together with what they perceive themselves to use and what they would value in further training. METHODS: We observed learning episodes for students and doctors-in-training in medical and surgical clinics. A questionnaire on outpatient teaching was also sent to consultant doctors and surgeons. This was based on these observations and focus groups with students and doctors-in-training. RESULTS: There was an overall survey response rate of 62% (194/311). The dominant forms of learning we observed were 'arms-length' supervision for doctors-in-training and 'modelling' for students. Only 7% of learning episodes involved a doctor-in-training doing something under direct supervision. In contrast to the observation results, consultants considered that students and doctors-in-training received a lot of direct supervision and interaction. For example, 45% considered that doctors-in-training 'may see patients with me in a joint consultation'. Only 30% of respondents would be interested in staff development in learning in outpatient clinics. CONCLUSIONS: Although consultants reported that they frequently used an active approach to learning in outpatient clinics, modelling was used predominantly for students and arms-length supervision was used for doctors-in-training.

Involving medical students in staff development activities.

Staff development is seen as a key aspect of medical education and many teachers/tutors are now required to attend some formal development activity. However, most workshops do not involve the student. In Manchester, the philosophy is to actively involve students in all aspects of the undergraduate course including delivery and evaluation. The authors have also worked with students in staff development. This predominantly descriptive article sets out some of the ways in which students have been involved in staff development, including co-facilitating sessions, role-play and discussion groups. Feedback from 10 workshops was analysed, which showed that 76% of the staff found the student involvement to be the most useful aspect of the workshops, particularly as it allowed staff to engage students in discussion about the course. Both these results and informal feedback from participants suggest that involvement of students is highly valued. Feedback from student volunteers also suggests that they gain from the experience.

Perceptions of how well graduates are prepared for the role of pre-registration house officer: a comparison of outcomes from a traditional and an integrated PBL curriculum.

INTRODUCTION: Most medical schools in the UK have been engaged in major curriculum reform based on their premises of what might improve undergraduate medical education. In 1994 the course at the medical school of the University of Manchester changed to an integrated course using problem-based learning throughout and with increased emphasis on community-based medical education. This study explores whether the new curriculum has produced any differences in perceptions of how well graduates are prepared for the role of pre-registration house officer. METHODS: A postal questionnaire was used to survey 1998 Manchester graduates (traditional course) and 1999 Manchester graduates (new course), three months into their first pre-registration house officer placement. A similar questionnaire was sent to the educational supervisors who were supervising the graduates. The questionnaire was designed to measure perceptions of levels of preparedness for the role of pre-registration house officer, using a list of broad areas of competence and specific skills listed in the General Medical Council's 'The New Doctor'. RESULTS: Graduates rated the new course significantly more effective for 12 of the 19 broad competences and eight of the 13 specific skills that were listed. The 'new' graduates rated their understanding of disease processes lower than the 'traditional' graduates, but there was no difference in the ratings given by the educational supervisors for this. Overall the educational supervisors rated the new course as better preparing graduates in five of the competences. CONCLUSIONS: Overall, the evaluation shows that a major change in curriculum approach has changed the profile of the perceived preparedness of graduates for entering professional practice.

A qualitative study of the attitudes to teamwork of graduates from a traditional and an integrated undergraduate medical course.

INTRODUCTION: In 1994 the University of Manchester medical school introduced an integrated undergraduate course using problem-based learning throughout. This study explores differences between the new and old (traditional) course graduates' attitudes to, and conceptualization of, teamwork. METHODS: Semi-structured interviews were conducted with 24 graduates of the traditional course (graduating in 1998) and 23 from the new course (1999 graduates), representing approximately 14% of graduates from each cohort. Theories were then developed from concepts emerging from the data. RESULTS: The new course graduates (NCGs) had a broader view of members of a health professional team. The NCGs believed that the medical team should provide support and were more comfortable consulting them when faced with problems. CONCLUSIONS: The new curriculum has had some impact on conceptualization and attitude to teamwork. However, further development is required if graduates are to see themselves as part of a multi-professional team.

Student access and use of IT during general practice attachments.

As universities change the delivery of their medical undergraduate courses, many have started to develop the community as a learning resource. In Manchester, part of the rationale behind problem-based learning is that students become familiar with the tools for finding information. Within the medical school and its main teaching hospitals, students have access to varied information resources using IT. These are often less available from off-campus sites and particularly within general practice. Even where available, students may not use them. Initial evaluation suggests that IT facilities are particularly useful for students who have longer placements. However, students prioritize their use of time in the community and furnishing general practices with a computer and other IT equipment does not, in itself, promote use by the students unless there is a perceived need. Training and support within the practice needs to be undertaken to enhance the learning environment for the undergraduates.

Does a new undergraduate curriculum based on Tomorrow's Doctors prepare house officers better for their first post? A qualitative study of the views of pre-registration house officers using critical incidents.

INTRODUCTION: In 1994 Manchester University introduced an integrated undergraduate medical course using problem-based learning (PBL) throughout. The study reported here explored whether there were any differences between the new course graduates (NCGs) and the traditional course graduates (TCGs) in the types of scenarios they recalled as 'critical incidents', or challenging cases, while working as pre-registration house officers (PRHOs). The focus is on differences rather than causal links. METHOD: We used semistructured interviews to generate our data. Twenty-four traditional course graduates and 23 new course graduates were interviewed approximately 3 months after starting their first PRHO placement. RESULTS: We identified 4 types of critical incidents relating to: clinical practice; limitations of competence; emotional involvement; and communication. Traditional course graduates reported difficulties in making patient management decisions, whereas the NCGs were better at dealing with uncertainty, knowing their limits and asserting their rights for support. Communication difficulties and coping with emotional involvement were common across both groups of graduates and hence remain problems in relation to being prepared for the role of a PRHO. CONCLUSIONS: Graduates of the new, integrated curriculum seemed to be much better at dealing with uncertainty, knowing their personal limits and asserting their rights for support when they felt these limits had been reached. Communication difficulties and emotional involvement remain major factors in the transition from student to PRHO.