Application of TraDIS to define the core essential genome of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the major cause of bacterial gastroenteritis. As there is no effective vaccine, combined with the rapid increase in antimicrobial resistant strains, there is a need to identify new targets for intervention. Essential genes are those that are necessary for growth and/or survival, making these attractive targets. In this study, comprehensive transposon mutant libraries were created in six C. jejuni strains, four C. coli strains and one C. lari and C. hyointestinalis strain, allowing for those genes that cannot tolerate a transposon insertion being called as essential. Comparison of essential gene lists using core genome analysis can highlight those genes which are common across multiple strains and/or species. Comparison of C. jejuni and C. coli, the two species that cause the most disease, identified 316 essential genes. Genes of interest highlighted members of the purine pathway being essential for C. jejuni whilst also finding that a functional potassium uptake system is essential. Protein-protein interaction networks using these essential gene lists also highlighted proteins in the purine pathway being major 'hub' proteins which have a large number of interactors across the network. When adding in two more species (C. lari and C. hyointestinalis) the essential gene list reduces to 261. Within these 261 essential genes, there are many genes that have been found to be essential in other bacteria. These include htrB and PEB4, which have previously been found as core virulence genes across Campylobacter species in other studies. There were 21 genes which have no known function with eight of these being associated with the membrane. These surface-associated essential genes may provide attractive targets. The essential gene lists presented will help to prioritise targets for the development of novel therapeutic and preventative interventions.

A virtuous cycle of co-production: Reflections from a community priority-setting exercise.

INTRODUCTION: Co-production is gaining increasing recognition as a good way of facilitating collaboration among different stakeholders, including members of the public. However, it remains an ambiguous concept as there is no definitive or universal model of co-production or clarity on what constitutes a good co-production approach. This paper draws on the reflections of the academic researchers, practitioners and public advisors involved in co-producing a priority-setting exercise. The exercise was conducted by the Primary and Community Health Services (PCHS) Theme of the National Institute for Health and Care Research Applied Research Collaboration for Kent, Surrey and Sussex (NIHR ARC KSS). METHODS: We collected data through written and verbal reflections from seven collaborators involved in the PCHS priority-setting exercise. We used Gibbs' model of reflection to guide the data collection. We then analysed the data through an inductive, reflexive thematic analysis. RESULTS: A common thread through our reflections was the concept of 'sharing'. Although co-production is inherently shared, we used the virtuous cycle to illustrate a sequence of sharing concepts during the research cycle, which provides the underpinnings of positive co-production outcomes. We identified six themes to denote the iterative process of a shared approach within the virtuous cycle: shared values, shared understanding, shared power, shared responsibilities, shared ownership and positive outcomes. CONCLUSION: Our results present a virtuous cycle of co-production, which furthers the conceptual underpinnings of co-production. Through our reflections, we propose that positive co-production outcomes require foundations of shared values and a shared understanding of co-production as a concept. These foundations facilitate a process of shared power, shared responsibilities and shared ownership. We argue that when these elements are present in a co-production exercise, there is a greater potential for implementable outcomes in the communities in which the research serves and the empowerment of collaborators involved in the co-production process. PUBLIC MEMBERS' CONTRIBUTIONS: Three members of the public who are public advisors in the NIHR ARC KSS were involved in the priority-setting exercise that informed this paper. The public advisors were involved in the design of the priority-setting exercise and supported participants' recruitment. They also co-facilitated the focus groups during data collection and were involved in the data analysis, interpretation and preparation of the priority-setting report. For this current manuscript, two of them are co-authors. They provided reflections and contributed to the writing and reviewing of this manuscript.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Cry75Aa (Mpp75Aa) Insecticidal Proteins for Controlling the Western Corn Rootworm, Diabrotica virgifera virgifera, (Coleoptera: Chrysomelidae), Isolated from the Insect Pathogenic Bacteria Brevibacillus laterosporus.

This study describes three closely related proteins, cloned from Brevibacillus laterosporus strains, that are lethal upon feeding to Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR). Mpp75Aa1, Mpp75Aa2 and Mpp75Aa3 were toxic to WCR larvae when fed purified protein. Transgenic plants expressing each mMpp75Aa protein were protected from feeding damage and showed significant reduction in adult emergence from infested plants by both susceptible and Cry3Bb1 and Cry34Ab1/Cry35Ab1-resistant WCR. These results demonstrate that proteins from B. laterosporus are as efficacious as the well-known Bacillus thuringiensis (Bt) insecticidal proteins in controlling major insect pests such as WCR. The deployment of transgenic maize expressing mMpp75Aa along with other active molecules lacking cross-resistance have the potential to be a useful tool for control of WCR populations resistant to current Bt traits.IMPORTANCE Insects feeding on roots of crops can damage the plant roots resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize the western corn rootworm (WCR) can cause severe damage to the roots resulting in significant economic loss for farmers. Genetically modified (GM) expressing Bacillus thuringiensis (Bt) insect control proteins, has provided a solution for control of these pests. In recent years populations of WCR resistant to the Bt proteins in commercial GM maize have emerged. There is a need to develop new insecticidal traits for the control of WCR populations resistant to current commercial traits. New proteins with commercial level efficacy on WCR from sources other than Bt are becoming more critical. The Mpp75Aa proteins, from B. laterosporus, when expressed in maize, are efficacious against the resistant populations of WCR and have the potential to provide solutions for control of resistant WCR.

O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli.

The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to "last-resort" antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.

Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.

Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection.

Opportunistic infections caused by Pseudomonas aeruginosa can be acute or chronic. While acute infections often spread rapidly and can cause tissue damage and sepsis with high mortality rates, chronic infections can persist for weeks, months, or years in the face of intensive clinical intervention. Remarkably, this diverse infectious capability is not accompanied by extensive variation in genomic content, suggesting that the genetic capacity to be an acute or a chronic pathogen is present in most P. aeruginosa strains. To investigate the genetic requirements for acute and chronic pathogenesis in P. aeruginosa infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in murine models of burn and non-diabetic chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for colonization and persistence in these infections. Specifically, we found that long-chain fatty acids represent a major carbon source in both chronic and acute wounds, and P. aeruginosa must biosynthesize purines, several amino acids, and most cofactors during infection. In addition, we determined that P. aeruginosa requires chemotactic flagellar motility for fitness and virulence in acute burn wound infections, but not in non-diabetic chronic wound infections. Our results provide novel insight into the genetic requirements for acute and chronic P. aeruginosa wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence.

A human gut microbial gene catalogue established by metagenomic sequencing.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response.

Pathogenicity of Pseudomonas aeruginosa, a major cause of many acute and chronic human infections, is determined by tightly regulated expression of multiple virulence factors. Quorum sensing (QS) controls expression of many of these pathogenic determinants. Previous microarray studies have shown that the AmpC ß-lactamase regulator AmpR, a member of the LysR family of transcription factors, also controls non-ß-lactam resistance and multiple virulence mechanisms. Using RNA-Seq and complementary assays, this study further expands the AmpR regulon to include diverse processes such as oxidative stress, heat shock and iron uptake. Importantly, AmpR affects many of these phenotypes, in part, by regulating expression of non-coding RNAs such as rgP32, asRgsA, asPrrF1 and rgRsmZ. AmpR positively regulates expression of the major QS regulators LasR, RhlR and MvfR, and genes of the Pseudomonas quinolone system. Chromatin immunoprecipitation (ChIP)-Seq and ChIP-quantitative real-time polymerase chain reaction studies show that AmpR binds to the ampC promoter both in the absence and presence of ß-lactams. In addition, AmpR directly binds the lasR promoter, encoding the QS master regulator. Comparison of the AmpR-binding sequences from the transcriptome and ChIP-Seq analyses identified an AT-rich consensus-binding motif. This study further attests to the role of AmpR in regulating virulence and physiological processes in P. aeruginosa.

A genomewide mutagenesis screen identifies multiple genes contributing to Vi capsular expression in Salmonella enterica serovar Typhi.

A transposon-based, genomewide mutagenesis screen exploiting the killing activity of a lytic ViII bacteriophage was used to identify Salmonella enterica serovar Typhi genes that contribute to Vi polysaccharide capsule expression. Genes enriched in the screen included those within the viaB locus (tviABCDE and vexABCDE) as well as oxyR, barA/sirA, and yrfF, which have not previously been associated with Vi expression. The role of these genes in Vi expression was confirmed by constructing defined null mutant derivatives of S. Typhi, and these were negative for Vi expression as determined by agglutination assays with Vi-specific sera or susceptibility to Vi-targeting bacteriophages. Transcriptome analysis confirmed a reduction in expression from the viaB locus in these S. Typhi mutant derivatives and defined regulatory networks associated with Vi expression.

An epigenetic switch mediates bistable expression of the type 1 pilus genes in Streptococcus pneumoniae.

Expression of the pneumococcal type 1 pilus is bistable and positively regulated by the transcription factor RlrA. RlrA is also known to positively control its own expression. Here we present evidence that bistable expression of the type 1 pilus is mediated by the positive-feedback loop controlling rlrA expression.

Epigenetic control of virulence gene expression in Pseudomonas aeruginosa by a LysR-type transcription regulator.

Phenotypic variation within an isogenic bacterial population is thought to ensure the survival of a subset of cells in adverse conditions. The opportunistic pathogen Pseudomonas aeruginosa variably expresses several phenotypes, including antibiotic resistance, biofilm formation, and the production of CupA fimbriae. Here we describe a previously unidentified bistable switch in P. aeruginosa. This switch controls the expression of a diverse set of genes, including aprA, which encodes the secreted virulence factor alkaline protease. We present evidence that bistable expression of PA2432, herein named bexR (bistable expression regulator), which encodes a LysR-type transcription regulator, controls this switch. In particular, using DNA microarrays, quantitative RT-PCR analysis, chromatin immunoprecipitation, and reporter gene fusions, we identify genes directly under the control of BexR and show that these genes are bistably expressed. Furthermore, we show that bexR is itself bistably expressed and positively autoregulated. Finally, using single-cell analyses of a GFP reporter fusion, we present evidence that positive autoregulation of bexR is necessary for bistable expression of the BexR regulon. Our findings suggest that a positive feedback loop involving a LysR-type transcription regulator serves as the basis for an epigenetic switch that controls virulence gene expression in P. aeruginosa.

Expression of the type 1 pneumococcal pilus is bistable and negatively regulated by the structural component RrgA.

The pneumococcal type 1 pilus, which is present in 25 to 30% of clinical isolates, has been associated with increased adherence and inflammatory responses and is being evaluated as a potential vaccine candidate. Here we show that expression of the pilus is bistable as a result of the molecular interaction between the transcription activator RrlA and a structural component of the pilus called RrgA. Sampling various clinical pneumococcal isolates that harbor the type 1 pilus-encoding islet, we show that distinct populations of cells can be identified with either undetectable or prominent pilus expression. When these two populations are separated and regrown in liquid medium, they are phenotypically different: the nonexpressing population reverts to the previous bimodal distribution, whereas the expressing population retains the same high level of pilus expression. Controlled exogenous expression of the regulatory pilus gene rlrA in a strain from which the endogenous version has been deleted increases pilus expression steadily, suggesting that the bistable expression of the pilus observed in wild-type cells is dependent on the native rlrA promoter. Finally, we demonstrate that RrgA is a negative regulator of pilus expression and that this repression is likely mediated through direct interaction with RlrA. We conclude that type 1 pilus expression in pneumococcus exhibits a bistable phenotype, which is dependent upon the molecular interplay between the RlrA and RrgA proteins. We suggest that this flexibility in expression may assist adaptation to a range of immune conditions, such as evasion of antipilus antibodies, within potential hosts.

H-NS family members function coordinately in an opportunistic pathogen.

The histone-like nucleoid structuring protein, H-NS, is a prominent global regulator of gene expression. Many Gram-negative bacteria contain multiple members of the H-NS family of proteins. Thus, a key question is whether H-NS family members have overlapping or distinct functions. To address this question we performed genome-wide location analyses with MvaT and MvaU, the two H-NS family members present in Pseudomonas aeruginosa. We show that MvaT and MvaU bind the same chromosomal regions, coregulating the expression of approximately 350 target genes. We show further that like H-NS in enteric bacteria, which functions as a transcriptional silencer of foreign DNA by binding to AT-rich elements, MvaT and MvaU bind preferentially to AT-rich regions of the chromosome. Our findings establish that H-NS paralogs can function coordinately to regulate expression of the same set of target genes, and suggest that MvaT and MvaU are involved in silencing foreign DNA elements in P. aeruginosa.

The level of willingness to evacuate among older adults.

The issues of rising numbers of disasters, overwhelming increases in number of older adults, and historically flawed evacuations present real challenges. During the next two decades, the number of American baby boomers, who turn 65, will increase by 40%. As evidenced by recent disasters, the imperfections and vulnerabilities of flawed evacuations for older adults are still present. This study examined the level of willingness to evacuate among older adults in the event of a disaster. Despite the extensive literature on disasters and evacuation, a significant question regarding evacuation and older adults has not been addressed. The study addressed the following concern: What is the level of willingness among older adults to evacuate when asked to do so by emergency management officials? The sample population consisted of 765 voluntary participants aged 60 years and older from 30 senior/community centers within seven counties within a midwestern state. A group administered survey (The Disaster Evacuation Survey) included a total of 15 questions. The findings revealed that older adults are more likely to comply with a mandatory evacuation order. Important practical implications for emergency officials responding to vulnerable older adults in disaster situations are also provided.

ORFograph: search for novel insecticidal protein genes in genomic and metagenomic assembly graphs.

BACKGROUND: Since the prolonged use of insecticidal proteins has led to toxin resistance, it is important to search for novel insecticidal protein genes (IPGs) that are effective in controlling resistant insect populations. IPGs are usually encoded in the genomes of entomopathogenic bacteria, especially in large plasmids in strains of the ubiquitous soil bacteria, Bacillus thuringiensis (Bt). Since there are often multiple similar IPGs encoded by such plasmids, their assemblies are typically fragmented and many IPGs are scattered through multiple contigs. As a result, existing gene prediction tools (that analyze individual contigs) typically predict partial rather than complete IPGs, making it difficult to conduct downstream IPG engineering efforts in agricultural genomics. METHODS: Although it is difficult to assemble IPGs in a single contig, the structure of the genome assembly graph often provides clues on how to combine multiple contigs into segments encoding a single IPG. RESULTS: We describe ORFograph, a pipeline for predicting IPGs in assembly graphs, benchmark it on (meta)genomic datasets, and discover nearly a hundred novel IPGs. This work shows that graph-aware gene prediction tools enable the discovery of greater diversity of IPGs from (meta)genomes. CONCLUSIONS: We demonstrated that analysis of the assembly graphs reveals novel candidate IPGs. ORFograph identified both already known genes "hidden" in assembly graphs and potential novel IPGs that evaded existing tools for IPG identification. As ORFograph is fast, one could imagine a pipeline that processes many (meta)genomic assembly graphs to identify even more novel IPGs for phenotypic testing than would previously be inaccessible by traditional gene-finding methods. While here we demonstrated the results of ORFograph only for IPGs, the proposed approach can be generalized to any class of genes. Video abstract.

High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials.

The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

Perception of Falls and Confidence in Self-Management of Falls among Older Adults.

Objectives: Fall preventive programs aim to reduce risks for mortality from fall-related injuries among older adults. However, the covariation between personal perceptions of falls and factors and confidence of self-management in falls (CSMoF) is still under-studied despite its importance to fall prevention. We aimed to investigate the relative contribution of CSMoF in relation to fall risk self-perceptions while controlling for demographics and self-reported health and functioning. Method: Participants were 691 older adults recruited from Area Agency on Aging at Arlington, Texas (females = 76.1%, mean age = 76.23, SD = 6.44, with chronic condition = 79.5%). They completed measures of physical functioning, CSMoF, fall risk perceptions and fear of falls. Results: Regression analyses indicated that fear of fall was the most predictive factor of CSMoF among older persons, accounting for about 25% of the variance. Physical function measures of age, chronic illnesses of metabolism, sensory impairment, and health status were also significant predictors of the CSMoF, but to a lesser extent than fear of falls and fall perceptions. The interaction of perception of falls and fall experience attenuated CSMoF, with physical functioning limitations. Conclusion: The joint effects of perception of falls and fear of falls likely explain CSMoF among older adults more than physical functional indicators. Fall prevention programs for older adults should prioritize to address modifiable subjective factors of fall perceptions, fear of falls, and CSMoF across health and functioning statuses.

The Essential Genome of Escherichia coli K-12.

Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCE Incentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in Escherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for the E. coli model laboratory organism. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.

An exploratory study of the needs of staff who care for offenders with a diagnosis of personality disorder.

People with a diagnosis of personality disorder who are considered a risk to others are a current concern for public services, government and society. Healthcare provision for this group has recently increased, making it important to begin to learn about the needs of staff working in such a challenging area. An exploratory interview study with staff working in a medium secure unit for offenders with a diagnosis of personality disorder (Unit Z) is presented. Interviews were line-coded and analysed according to the grounded theory method. A model was developed from higher order categories. This related key contextual factors to areas of concern for staff. The key contextual factors were: 'complexity of the task' and 'tension in the relationship with outside'. The areas of concern were: 'desire for more meaningful contact', 'contradictory attitude towards openness', 'feeling physically safe but emotionally vulnerable', 'ambivalence towards structure and control' and 'emphasis on staff relationships'. 'Risk of isolation' formed a core category and described Unit Z staff's pervading sense of distance from the outside world. These categories are described with supporting quotations and discussed in relation to the existing literature. Their implications for clinical practice and the development of services are considered.