Elements of person-centred diagnostic imaging care in low-and middle-income countries: A systematic review.

INTRODUCTION: Diagnostic imaging professionals are trained to deliver safe and high-quality person-centred radiographic diagnostic imaging care. The term person-centred care has been described as a confused concept without a unified definition. This systematic review identified the elements that have been used to measure person-centred care in diagnostic imaging in low- and middle-income countries (LMICs). METHODS: A systematic review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis statement. Embase, MEDLINE and Cochrane library were searched. Bias was assessed using the Critical Appraisal Skill Programme and Mixed Method Appraisal Tool. A narrative synthesis guided by the Picker Principles of person-centred care was undertaken. RESULTS: Of the 4482 articles identified, 26 articles were included. The studies were from 12 LMICs. Synthesis of the literature generated six themes, namely access to high quality and safe diagnostic imaging care, effective communication and shared diagnostic imaging decision making, suitable diagnostic imaging environment for physical comfort, respectful and compassionate diagnostic radiographers, effective coordination of diagnostic imaging care process, and family and friends' involvement in diagnostic imaging care. CONCLUSION: Medical imaging facilities in most LMICs continue to struggle with issues of access, safety, quality, and responsiveness to the needs of patients. The need for innovative person-centred diagnostic imaging care interventions in LMICs has become urgent. IMPLICATIONS FOR PRACTICE: If diagnostic imaging services in LMICs are to move beyond the current models of limited person-centred access to care, a greater focus on systems thinking is required. It is imperative to involve all stakeholders, not only patients and radiographers, but also policymakers whose works impact on equitable access to diagnostic imaging services.

Exploring UK sonographers' views on the use of professional supervision in clinical practice - Stage one findings of a mixed method study.

INTRODUCTION: Professional Supervision has been described across multiple professional groups, however to date, minimal research has been conducted exploring the use of professional supervision within the United Kingdom (UK) sonographer workforce. METHODS: An online self-administered survey was conducted to explore UK sonographers views on the use of professional supervision in practice. The survey was open to sonographers, consultant or clinical specialist sonographers, ultrasound managers and professional body officers. Multiple choice questions were utilised to obtain quantitative data on the provision of support mechanisms, with free text questions allowing qualitative data to be elicited further to explore thoughts of participants. RESULTS: A total of 112 participants completed the survey in full and response rates varied across the subgroups. Varying support mechanisms were in place for sonographers. However only 55.4 % of sonographers felt supported in the clinical workplace. Thematic analysis of qualitative data highlighted that workload pressures, staffing and retention of sonographers, were key concerns that professional supervision could improve. It was highlighted that time to undertake professional supervision could be challenging, however if training for professional supervision was in place then this could provide improved quality of care and staff support. CONCLUSION: Participants highlighted the challenges faced by UK sonographers and the positive impact that professional supervision could have on retention and staff support. There are limited support mechanisms in place for UK sonographers and this is impacting on how participants felt they were being supported in the workplace. Stage 2 of this research project will explore sonographers' views in more detail. IMPLICATIONS FOR PRACTICE: The approach to support mechanisms for sonographers should be considered to support improvement of professional wellbeing and retention of the sonographic workforce.

Promotion of research culture among radiographers in one UK NHS trust through journal club activities - An autoethnographic study.

INTRODUCTION: The poor research culture within the radiography profession can be addressed through initiatives such journal club activities. The role of a research radiographer is best suited to drive achievement of journal club outcomes for research culture improvement; however, the cultural environment of healthcare providers presents challenges. This is an autoethnographic account of a research radiographer in promoting research culture among diagnostic radiographers in a single UK NHS trust through journal club activities. METHODS: The study adopts an analytical autoethnography methodology to critically analyse reflective accounts of the research radiographer's experiences and the interplay with the cultural environment in which the experiences occurred. The reflective accounts are supported by locally collected data during a 10-month period of the journal club and published literature. RESULTS: The establishment of the journal club received encouraging support from the senior management, university academics, library services and radiography professionals. There are initial signs of research culture improvement among participants of the journal club as observed through engagement in research related activities. However, cultural challenges including lack of time to explore gaps in research evidence, and prioritisation of clinical duties over research related activities, may have affected the achievement of expected outcomes of the club. CONCLUSION: The research radiographer is well placed to encourage research culture within the clinical imaging department through targeted initiatives such as journal clubs. The long-term contributions of such initiative to departmental efficiency and quality service delivery should encourage optimum support to actualize expected outcomes. IMPLICATIONS FOR PRACTICE: Encouragement of journal clubs as a means of improving research culture within clinical radiography teams, driven by research radiographers. Encouragement of management support for the achievement of set outcomes of journal clubs.

Post-acute COVID syndrome (long COVID): What should radiographers know and the potential impact for imaging services.

OBJECTIVES: The COVID-19 pandemic caused an unprecedented health crisis resulting in over 6 million deaths worldwide, a figure, which continues to grow. In addition to the excess mortality, there are individuals who recovered from the acute stages, but suffered long-term changes in their health post COVID-19, commonly referred to as long COVID. It is estimated there are currently 1.8 million long COVID sufferers by May 2022 in the UK alone. The aim of this narrative literature review is to explore the signs, symptoms and diagnosis of long COVID and the potential impact on imaging services. KEY FINDINGS: Long COVID is estimated to occur in 9.5% of those with two doses of vaccination and 14.6% if those with a single dose or no vaccination. Long COVID is defined by ongoing symptoms lasting for 12 or more weeks post acute infection. Symptoms are associated with reductions in the quality of daily life and may involve multisystem manifestations or present as a single symptom. CONCLUSION: The full impact of long COVID on imaging services is yet to be realised, but there is likely to be significant increased demand for imaging, particularly in CT for the assessment of lung disease. Educators will need to include aspects related to long COVID pathophysiology and imaging presentations in curricula, underpinned by the rapidly evolving evidence base. IMPLICATIONS FOR PRACTICE: Symptoms relating to long COVID are likely to become a common reason for imaging, with a particular burden on Computed Tomography services. Planning, education and updating protocols in line with a rapidly emerging evidence base is going to be essential.

Is there a role for professional supervision in supporting the professional and personal wellbeing of the sonographic workforce? A literature review.

OBJECTIVES: The sonographer workforce in the UK is under pressure due to chronic staff shortages and increased service demands. The Health and Care Professions Council and the Society of Radiographers both advocate for the use of professional supervision to support wellbeing and development, however the use of professional supervision is not widespread within the sonographer workforce. The aim of this literature review was to explore the evidence around the use of professional supervision and how this could support sonographer wellbeing. KEY FINDINGS: Professional supervision was found to be of importance for providing emotional support which can lead to increased job satisfaction, lower levels of burnout and subsequently impact on retention of the workforce. Professional supervision supported personal development and therefore increased quality of patient care, allowing professionals to discuss evidence-based care, policies and practice development through reflection. Whilst important for emotional and professional support, this review found that there are conflicting pressures which can impact the effectiveness of supervision including the supervisory relationship itself and time and organisational pressures. Demands on the workforce made a significant impact on the availability of quality clinical supervision. CONCLUSION: Professional supervision has an important role in supporting the sonography workforce and enable increased wellbeing and emotional support. There are clear benefits to undertaking professional supervision to support the workforce however there are competing demands which may affect the effectiveness of professional supervision. IMPLICATIONS FOR PRACTICE: There is limited international research on the use of professional supervision in the radiographer and sonographer workforce. There are clear benefits for supporting professional wellbeing and development under a structured professional supervision framework however within the United Kingdom there is a significant gap in the literature where further research is required.

First year student radiographers' perceptions of a one-week simulation-based education package designed to increase clinical placement capacity.

INTRODUCTION: The radiography workforce is short-staffed and under increasing pressure to meet service pressures. Combined with the impact of Covid-19, where student face-to-face clinical time was abruptly halted for safety, there is cause to change the pedagogical approach to teaching diagnostic radiography to students, increasing capacity and ensuring the continuance of qualifying radiographers to support the profession. This paper shares the perceptions of first year student radiographers on a one-week simulation-based education package designed to replace one week of clinical placement experience. METHODS: Two cohorts of first-year radiography students engaged in a one-week simulation-based education package. Simulations increased in complexity throughout the week and included conventional imaging techniques, mobile and theatre radiography, and cross-sectional imaging. Thirty-six students consented to the thematic analysis of their reflective blogs. RESULTS: Five themes emerged from the data: feeling anxious, understanding and skill development, building confidence, communication, and patient-centred care. CONCLUSION: The simulation package had a positive impact on students learning, no matter the stage at which it was incorporated into their clinical placement block. Students engaged well with the activities and saw value in the experience. The findings indicate that the simulation-based education package is a suitable replacement for one week of clinical placement, supporting skills development in students and providing increased placement capacity. IMPLICATIONS FOR PRACTICE: A successful, engaging simulation-based education package is presented, which first year student radiographers perceived as a suitable replacement for one-week of clinical placement. Further research into the acceptability of use of simulation-based education packages in second- and third-year student radiographers would be a useful next step.

Backbone assignments, and effect of Asn deamidation, of the N-terminal region of the partitioning protein IncC1 from the plasmid RK2.

IncC from the low-copy number plasmid RK2, is a member of the ParA family of proteins required for partitioning DNA in many bacteria and plasmids. It is an ATPase that binds DNA and its ParB protein partner, KorB. Together, the proteins move replicated DNA to appropriate cellular positions, so that each daughter cell inherits a copy on cell division. IncC from RK2 is expressed in two forms. IncC2 is homologous to bacterial ParA proteins, while IncC1 has an N-terminal extension of 105 amino acids and is similar in length to ParA homologues in other plasmids. We have been examining the role of this extension, here called IncC NTD. We present its backbone NMR chemical shift assignments and show that it is entirely intrinsically disordered. The assignments were achieved using C-detected, CON-based spectra, complemented by HNN spectra to obtain connectivities from three adjacent amino acids. We also observed evidence of deamidation of the protein at a GNGG sequence, to give isoAsp, giving 2 sets of peaks for residues up to 5 amino acids on either side of the modification. We have assigned resonances from around the position of modification for this form of the protein.

Patient centred care in diagnostic radiography (Part 3): Perceptions of student radiographers and radiography academics.

INTRODUCTION: Awareness is growing of the importance of patient centered care (PCC) in diagnostic radiography. PCC is embedded within professional body publications and guidance documents, but there is limited research evidence exploring the perceptions of student radiographers and radiography academics. METHODS: This paper reports Stage 1 and Stage 2 of the project from the perspective of radiography academic and student radiographer participants, and compares these to the perspectives of service users, clinical radiographers and radiography managers reported previously. Stage 1 used an online survey tool to gauge participant agreement with a series of attitudinal statements. Stage 2 used situational vignettes to promote discussion and debate about PCC approaches. RESULTS: Response rates to the Stage 1 survey were above the minimum threshold, with 50 responses from student radiographers and 38 responses from radiography academics. Stage 1 participants were asked to participate in Stage 2 on a voluntary basis. As with service users and service deliverers, care communication, event interactions and control over environment were the key influences on PCC. However, students highlighted differences between reported and observed levels of PCC. CONCLUSION: There is some way to go to embed PCC in diagnostic radiography practice. As impartial observers of radiography practice, student radiographers highlight the difference between service users and service deliverer's perceptions of PCC. Whilst the focus of clinical radiographers remains on efficiency, it is difficult for student radiographers to challenge the accepted norm. Role models are required to promote PCC behaviours and a holistic approach in radiography practice. IMPLICATIONS FOR PRACTICE: A package of educational support and audit tools will be made available to support both service deliverers and student radiographers to deliver PCC.

Patient centred care in diagnostic radiography (Part 1): Perceptions of service users and service deliverers.

INTRODUCTION: There is growing awareness of the importance of patient centered care (PCC) in health care. Within Radiography in the UK, elements of PCC are embedded within professional body publications and guidance documents. However, there is limited research evidence exploring whether perceptions of PCC are equivalent between those delivering (radiographers) and those experiencing (patient) care. This study aimed to address this gap by determining compatibility in perceptions of PCC between those using and those delivering radiography services. This is the first step in developing measurable indicators of PCC in diagnostic radiography. METHODS: A multi-method two stage approach was undertaken using survey and interview data collection techniques. Ethical approval was granted by University of Derby College of Health & Social Care Ethics committee. This paper reports Stage 1 of the study, the online, cross sectional survey. Participants were asked to indicate their level of agreement to a series of attitudinal statements using a 5-point Likert scale. Statements were paired, but not co-located to increase validity. Participants were invited to provide free text comments to supplement their responses. Stage 2 of the project is reported separately. RESULTS: Survey responses were received from all 3 participant subgroups. A minimum response rate of 30 participants per sub-group was set as a target. Response rates varied across subgroups, with only radiography managers failing to meet the expected response threshold. Wide disparity between perceptions of service users and those delivering radiography services on what constitutes high quality PCC was evident. CONCLUSION: It is evident that there is still work required to ensure parity between expectations of service users and deliverers on what constitutes high quality PCC. IMPLICATIONS FOR PRACTICE: Further work is required to identify measurable service delivery outcomes that represent PCC within radiographic practice.

Patient centred care in diagnostic radiography (Part 2): A qualitative study of the perceptions of service users and service deliverers.

INTRODUCTION: There is growing awareness of the importance of patient centred care (PCC) in health care. Within Radiography in the UK, elements of PCC are embedded within professional body publications and guidance documents, but there is limited research evidence exploring whether perceptions of PCC are equivalent between those delivering (radiographers) and those experiencing (patient) care. This study aimed to address this gap by determining compatibility in perceptions of PCC between those using and those delivering radiography services in order to develop measurable indicators of PCC. METHODS: This project was funded by the College of Radiographers Industry Partnership Scheme. Ethical approval was granted by the University of Derby College of Health & Social Care Ethics committee. This paper reports Stage 2 of the project, which was a series of focus groups and telephone interviews to enable deeper discussion and exploration of PCC. Situational vignettes were used to promote discussion and debate and encourage suggestions for PCC approaches. Audit tools to assess engagement with PCC were developed at individual and organisational level. RESULTS: Four focus groups and six telephone interviews were carried out in total. Focus groups were held in a variety of locations to promote attendance. Telephone interviews were used to capture participants who could not attend a focus group in person. Disparity between perceptions of service users and those delivering radiography services on what constitutes high quality PCC was evident. Perceived levels of care and the effectiveness of communication appeared to be the key influences on whether PCC was delivered. CONCLUSION: It is evident from the results of Stage 1 and Stage 2 that we have some way to go before we have parity in how care within diagnostic radiography is perceived, experienced and delivered. Audit tools and an educational toolkit are offered as ways to support increased PCC within diagnostic radiography practice. IMPLICATIONS FOR PRACTICE: Several service improvements and audit tools are offered to support the increased delivery of PCC.

Interactive virtual 3D models of renal cancer patient anatomies alter partial nephrectomy surgical planning decisions and increase surgeon confidence compared to volume-rendered images.

PURPOSE: To determine whether the interactive visualisation of patient-specific virtual 3D models of the renal anatomy influences the pre-operative decision-making process of urological surgeons for complex renal cancer operations. METHODS: Five historic renal cancer patient pre-operative computed tomography (CT) datasets were retrospectively selected based on RENAL nephrectomy score and variety of anatomy. Interactive virtual 3D models were generated for each dataset using image segmentation software and were made available for online visualisation and manipulation. Consultant urologists were invited to participate in the survey which consisted of CT and volume-rendered images (VRI) for the control arm, and CT with segmentation overlay and the virtual 3D model for the intervention arm. A questionnaire regarding anatomical structures, surgical approach, and confidence was administered. RESULTS: Twenty-five participants were recruited (54% response rate), with 19/25 having > 5 years of renal surgery experience. The median anatomical clarity score increased from 3 for the control to 5 for the intervention arm. A change in planned surgical approach was reported in 19% of cases. Virtual 3D models increased surgeon confidence in the surgical decisions in 4/5 patient datasets. There was a statistically significant improvement in surgeon opinion of the potential utility for decision-making purposes of virtual 3D models as compared to VRI at the multidisciplinary team meeting, theatre planning, and intra-operative stages. CONCLUSION: The use of pre-operative interactive virtual 3D models for surgery planning influences surgical decision-making. Further studies are needed to investigate if the use of these models changes renal cancer surgery outcomes.

Integrated multidisciplinary community service for chronic obstructive pulmonary disease reduces hospitalisations.

BACKGROUND: Hospitalisations for chronic obstructive pulmonary disease (COPD) exacerbation affect patient outcomes and healthcare costs. The long-term impact of an integrated COPD disease-management approach on hospitalisation remains controversial. AIM: The aim of this study was to evaluate whether a multidisciplinary community service reduces respiratory hospitalisations for COPD patients. METHODS: A total of 346 patients was followed for a mean duration of 27.3 months. The number of admissions, total bed days for respiratory (COPD exacerbation or pneumonia) or general medical causes and length of stay (LOS) per respiratory admission was compared before and after referral with the service. A secondary multivariate analysis examined which clinical parameters best predict benefit from such service. RESULTS: The total respiratory admission and hospital bed days after referral were reduced by 31% (288 vs 417, P < 0.001) and 40.4% (1637 vs 2746, P < 0.0001) respectively, compared with the equivalent duration prior. The average LOS for each respiratory admission was also significantly reduced after referral (6.61 vs 5.70, P = 0.02). Overall, 55% patients experienced a reduction in admission frequency and hospital days. The impact on admission frequency and hospital days was the greatest in those with an at least moderate disease (GOLD ≥2, odds ratio (OR): 3.2, 95% confidence interval (CI): 1.2, 8.9; P = 0.019) and those who completed pulmonary rehabilitation (PR) (OR: 1.7, 95% CI: 1.1, 2.8; P = 0.04). In contrast, general medical admissions increased, one-third attributable to a cardiovascular cause both before and after referral. CONCLUSIONS: The implementation of COPD multidisciplinary community service was associated with reduced respiratory hospitalisations in the long term. Patients with moderate or severe disease and who are able to complete PR are much more likely to benefit.

Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation.

Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase(+) dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103(+) and CD11b(+) DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP(+) DCs, corresponding to the CD103(+) DC subset, and XCR1-GFP(-) CD11c(+) cells, which include CD11b(+) DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103(+) and CD11b(+) DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.

A spatially-distributed computational model to quantify behaviour of contrast agents in MR perfusion imaging.

Contrast agent enhanced magnetic resonance (MR) perfusion imaging provides an early, non-invasive indication of defects in the coronary circulation. However, the large variation of contrast agent properties, physiological state and imaging protocols means that optimisation of image acquisition is difficult to achieve. This situation motivates the development of a computational framework that, in turn, enables the efficient mapping of this parameter space to provide valuable information for optimisation of perfusion imaging in the clinical context. For this purpose a single-compartment porous medium model of capillary blood flow is developed which is coupled with a scalar transport model, to characterise the behaviour of both blood-pool and freely-diffusive contrast agents characterised by their ability to diffuse through the capillary wall into the extra-cellular space. A parameter space study is performed on the nondimensionalised equations using a 2D model for both healthy and diseased myocardium, examining the sensitivity of system behaviour to Peclet number, Damköhler number (Da), diffusivity ratio and fluid porosity. Assuming a linear MR signal response model, sample concentration time series data are calculated, and the sensitivity of clinically-relevant properties of these signals to the model parameters is quantified. Both upslope and peak values display significant non-monotonic behaviour with regard to the Damköhler number, with these properties showing a high degree of sensitivity in the parameter range relevant to contrast agents currently in use. However, the results suggest that signal upslope is the more robust and discerning metric for perfusion quantification, in particular for correlating with perfusion defect size. Finally, the results were examined in the context of nonlinear signal response, flow quantification via Fermi deconvolution and perfusion reserve index, which demonstrated that there is no single best set of contrast agent parameters, instead the contrast agents should be tailored to the specific imaging protocol and post-processing method to be used.

A computationally efficient framework for the simulation of cardiac perfusion using a multi-compartment Darcy porous-media flow model.

We present a method to efficiently simulate coronary perfusion in subject-specific models of the heart within clinically relevant time frames. Perfusion is modelled as a Darcy porous-media flow, where the permeability tensor is derived from homogenization of an explicit anatomical representation of the vasculature. To account for the disparity in length scales present in the vascular network, in this study, this approach is further refined through the implementation of a multi-compartment medium where each compartment encapsulates the spatial scales in a certain range by using an effective permeability tensor. Neighbouring compartments then communicate through distributed sources and sinks, acting as volume fluxes. Although elegant from a modelling perspective, the full multi-compartment Darcy system is computationally expensive to solve. We therefore enhance computational efficiency of this model by reducing the N-compartment system of Darcy equations to N pressure equations, and N subsequent projection problems to recover the Darcy velocity. The resulting 'reduced' Darcy formulation leads to a dramatic reduction in algebraic-system size and is therefore computationally cheaper to solve than the full multi-compartment Darcy system. A comparison of the reduced and the full formulation in terms of solution time and memory usage clearly highlights the superior performance of the reduced formulation. Moreover, the implementation of flux and, specifically, impermeable boundary conditions on arbitrarily curved boundaries such as epicardium and endocardium is straightforward in contrast to the full Darcy formulation. Finally, to demonstrate the applicability of our methodology to a personalized model and its solvability in clinically relevant time frames, we simulate perfusion in a subject-specific model of the left ventricle.

A novel porous mechanical framework for modelling the interaction between coronary perfusion and myocardial mechanics.

The strong coupling between the flow in coronary vessels and the mechanical deformation of the myocardial tissue is a central feature of cardiac physiology and must therefore be accounted for by models of coronary perfusion. Currently available geometrically explicit vascular models fail to capture this interaction satisfactorily, are numerically intractable for whole organ simulations, and are difficult to parameterise in human contexts. To address these issues, in this study, a finite element formulation of an incompressible, poroelastic model of myocardial perfusion is presented. Using high-resolution ex vivo imaging data of the coronary tree, the permeability tensors of the porous medium were mapped onto a mesh of the corresponding left ventricular geometry. The resultant tensor field characterises not only the distinct perfusion regions that are observed in experimental data, but also the wide range of vascular length scales present in the coronary tree, through a multi-compartment porous model. Finite deformation mechanics are solved using a macroscopic constitutive law that defines the coupling between the fluid and solid phases of the porous medium. Results are presented for the perfusion of the left ventricle under passive inflation that show wall-stiffening associated with perfusion, and that show the significance of a non-hierarchical multi-compartment model within a particular perfusion territory.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle.

Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.