A Novel Biosensor Using Nanolithographically-Produced Submicron Optical Sources for the Study of Cell Adhesion and Chemotaxis

Cell adhesion and chemotaxis are two key factors determining cell behaviour and differentiation which are currently analysed by microscopic examination of the cell or membrane-associated fluorescence labels. These analyses are often slow, labour intensive and of limited informational content. This thesis describes the physical theory and experimental aspects of an optical method suitable for monitoring cell contact, adhesion to a surface and chemotaxis beyond the conventional limit of optical microscopy by means of a device that utilises both a plain bare surface and arrays of apertures nanolithographically-produced in the surface of a Surface Plasmon Resonance (SPR) sensor structure. Any minute vertical movement of the cell, within the near-field of the SPR active surface or actual cell/surface contact, creates intensity fluctuations, detectable in the far-field. This was demonstrated during experiments with non-apertured devices. (A video demonstrating the biological features of the device accompanies this thesis and may be obtained by contacting University of Plymouth's LRC.) The light scattered by each nanolithographically-produced aperture also fluctuates as a consequence of the cell approaching to within a few hundred nanometres of the aperture bearing surface and demonstrated detection of minute vertical movement on the surface of the apertured device. The combination of apertured and non-apertured detection results in a highly spatially-sensitive 3-dimensional sensor. Digitising the output from a CCD camera allows patterns of intensity fluctuation to be correlated with the contact and adhesion of individual cells on the active surface over a short period of time (2-3 minutes). Initial trials of an apertured device (diameter (^) « wavelength of incident light ( X ) ) carried out by our collaborating partners Drs R. Carr and S. Al-shukri at the Centre for Applied Microbiology and Research, Porton Down demonstrated that the use of apertures etched in a SPR metal surface produced a highly sensitive dielectric monitor, i.e. sensitive to very small changes in the refractive index of the micro-environment adjacent to the aperture. This was proposed as being of potentially great value in the development of extremely sensitive probes of dielectric particulates of sub-micron dimensions, i.e. biological macromolecules and supramolecular structures. Characterisation of the associated radiative and non-radiative evanescent fields on the surface of the device was conducted in order to gain a greater knowledge of the mechanisms by which the interactions between the cells adjacent to and in direct contact with the apertures and evanescent fields produced such significant intensity fluctuations in the results at CAMR. A combinational Scanning Probe Microscope was developed and used in Scanning Nearfield Optical Microscope and Photon Scanning Tunnelling Microscope modes of operation to detect the evanescent and radiative fields respectively. Detailed mapping of the radiative pattern in the near-field of the large apertures {<p » X) demonstrated a diffraction of approximately 25% of the Surface Plasmon Wave (SPW) either side of the centre of the aperture with the remainder being contained within the metal layer. Scattering at the second aperture interface, i.e. air/metal, was shown to be of a lower magnitude as a result of non-surface plasmon enhancement within the non-resonant aperture. Characterisation of the intensity profile of small apertures (^ < A) was beyond the scope of this project due to its limited time and finance and was not undertaken. A section in the conclusions is dedicated to giving a possible cause of the intensity profiles IV detected during the initial studies at CAMR with possible procedures required to verify and expand such work. In order to investigate the potential of the device in the biological environment, biological trials were carried out with collaborating establishments at Salisbury and Exeter and demonstrated that this dual sensing microscopic technique had great potential in the 3-dimensional monitoring of cell movement together with the capability of extending our knowledge of cell behaviour with the view to a system of rapid screening for tumour cells. This technique has produced real-time images of cell behaviour, which to our knowledge has not been previously seen before by any other microscopy technique. The finding of these trials are documented in this thesis with possible theories as to what the biological effects responsible for these results may possibly be. Future work into the verification of these effects and more biological trials and procedures are described in the hope that afler further work the device may be developed into a commercial and readily available scientific unit for use in the laboratory.FORCE Cancer Research Centre, Exeter and Centre for Applied Microbiology and Research, Porton Dow

Phoenix Y6

The mission of this project is to design and fabricate a vertical take-off and landing (VTOL) fixed-wing drone for use by firefighters and other emergency services. This vehicle will be designed for uses that include surveying wildfires, as well as spotting vehicular accidents, urban fires, and floods. Current drones available on the market are expensive or not designed specifically for emergency response. Our goal is to develop a working prototype of a vehicle that will be able to collect and relay important data such as live video and thermal images in addition to other measurements such as air velocity and humidity

Long-term outcomes of urinary tract infection (UTI) in Childhood (LUCI): protocol for an electronic record-linked cohort study

Funding This project has been funded by the Welsh Government through Health and Care Research Wales (project number 1068). Acknowledgments We acknowledge the support and input from Sarah Jones, our parent representative for the study. We are also grateful to the DUTY and EURICA participants for their agreement for continued use of their data for this study. The Centre for Trials Research receives funding from Health and Care Research Wales and Cancer Research UK. Wales Centre for Primary and Emergency Care Research (PRIME Centre Wales) receives funding from Health and Care Research Wales. The authors are supported by the Farr Institute CIPHER, funded by Arthritis Research UK, the British Heart Foundation, Cancer Research UK, the Economic and Social Research Council, the Engineering and Physical Sciences Research Council, the Medical Research Council, the National Institute of Health Research, the National Institute for Social Care and Health Research (Welsh Assembly Government), the Chief Scientist Office (Scottish Government Health Directorates), and the Wellcome Trust (MRC grant number MR/K006525/1) and the National Centre for Population Health and Wellbeing Research (NCPHWR). Ethics approval Ethics approval of the study has been given by the Research Ethics Committee for Wales (16/WA/0166) and the transfer and use of identifiable data has been approved by the Health Research Authority’s (HRA) Confidentiality Advisory Group (CAG) (16/CAG/0114).Peer reviewedPublisher PD

Opposing effects of agricultural intensification on two ecologically similar species

Brown hares and rabbits are widely distributed in agricultural landscapes across the UK, occupy similar habitats and have considerable dietary overlap. However, as agriculture in the UK has intensified, hares have declined and become a species of conservation concern while rabbits have become an increasing pest. An intensive study of hares, rabbits and the dynamics of pastures over two grazing seasons was undertaken, in order to understand the environmental factors associated with hare and rabbit abundance at field level. Linear mixed models were used to assess the environmental variables, in terms of the structure, nutritional components and effects of livestock grazing that are associated with the abundance of the two species. The models revealed that hares were negatively associated with grazing intensity and plant diversity, whereas rabbits showed the strongest associations with nutritional content of pastures, in particular fat, nitrogen and fibre content in forage, as well as a positive association with short grass swards. The data suggest that, at the field-scale intensification of pasture use may have contributed to declines in hares and increases in rabbits

Improving the diagnosis and treatment of urinary tract infection in young children in primary care:results from the ‘DUTY’ prospective diagnostic cohort study

PURPOSE Up to 50% of urinary tract infections (UTIs) in young children are missed in primary care. Urine culture is essential for diagnosis, but urine collection is often difficult. Our aim was to derive and internally validate a 2-step clinical rule using (1) symptoms and signs to select children for urine collection; and (2) symptoms, signs, and dipstick testing to guide antibiotic treatment. METHODS We recruited acutely unwell children aged under 5 years from 233 primary care sites across England and Wales. Index tests were parent-reported symptoms, clinician-reported signs, urine dipstick results, and clinician opinion of UTI likelihood (clinical diagnosis before dipstick and culture). The reference standard was microbiologically confirmed UTI cultured from a clean-catch urine sample. We calculated sensitivity, specificity, and area under the receiver operator characteristic (AUROC) curve of coefficient-based (graded severity) and points-based (dichotomized) symptom/sign logistic regression models, and we then internally validated the AUROC using bootstrapping. RESULTS Three thousand thirty-six children provided urine samples, and culture results were available for 2,740 (90%). Of these results, 60 (2.2%) were positive: the clinical diagnosis was 46.6% sensitive, with an AUROC of 0.77. Previous UTI, increasing pain/crying on passing urine, increasingly smelly urine, absence of severe cough, increasing clinician impression of severe illness, abdominal tenderness on examination, and normal findings on ear examination were associated with UTI. The validated coefficient- and points-based model AUROCs were 0.87 and 0.86, respectively, increasing to 0.90 and 0.90, respectively, by adding dipstick nitrites, leukocytes, and blood. CONCLUSIONS A clinical rule based on symptoms and signs is superior to clinician diagnosis and performs well for identifying young children for noninvasive urine sampling. Dipstick results add further diagnostic value for empiric antibiotic treatment

Prednisolone or pentoxifylline for alcoholic hepatitis

BACKGROUND: Alcoholic hepatitis is a clinical syndrome characterized by jaundice and liver impairment that occurs in patients with a history of heavy and prolonged alcohol use. The short-term mortality among patients with severe disease exceeds 30%. Prednisolone and pentoxifylline are both recommended for the treatment of severe alcoholic hepatitis, but uncertainty about their benefit persists.METHODS: We conducted a multicenter, double-blind, randomized trial with a 2-by-2 factorial design to evaluate the effect of treatment with prednisolone or pentoxifylline. The primary end point was mortality at 28 days. Secondary end points included death or liver transplantation at 90 days and at 1 year. Patients with a clinical diagnosis of alcoholic hepatitis and severe disease were randomly assigned to one of four groups: a group that received a pentoxifylline-matched placebo and a prednisolone-matched placebo, a group that received prednisolone and a pentoxifylline-matched placebo, a group that received pentoxifylline and a prednisolone-matched placebo, or a group that received both prednisolone and pentoxifylline.RESULTS: A total of 1103 patients underwent randomization, and data from 1053 were available for the primary end-point analysis. Mortality at 28 days was 17% (45 of 269 patients) in the placebo-placebo group, 14% (38 of 266 patients) in the prednisolone-placebo group, 19% (50 of 258 patients) in the pentoxifylline-placebo group, and 13% (35 of 260 patients) in the prednisolone-pentoxifylline group. The odds ratio for 28-day mortality with pentoxifylline was 1.07 (95% confidence interval [CI], 0.77 to 1.49; P=0.69), and that with prednisolone was 0.72 (95% CI, 0.52 to 1.01; P=0.06). At 90 days and at 1 year, there were no significant between-group differences. Serious infections occurred in 13% of the patients treated with prednisolone versus 7% of those who did not receive prednisolone (P=0.002).CONCLUSIONS: Pentoxifylline did not improve survival in patients with alcoholic hepatitis. Prednisolone was associated with a reduction in 28-day mortality that did not reach significance and with no improvement in outcomes at 90 days or 1 year. (Funded by the National Institute for Health Research Health Technology Assessment program; STOPAH EudraCT number, 2009-013897-42 , and Current Controlled Trials number, ISRCTN88782125 ).</p

Antibiotics for lower respiratory tract infection in children presenting in primary care (ARTIC-PC): the predictive value of molecular testing

Objectives This study aimed to assess whether the presence of bacteria or viruses in the upper airway of children presenting with uncomplicated lower respiratory tract infection (LRTI) predicts the benefit of antibiotics. Methods Children between 6 months and 12 years presenting to UK general practices with an acute LRTI were randomized to receive amoxicillin 50 mg/kg/d for 7 days or placebo. Children not randomized (ineligible or clinician/parental choice) could participate in a parallel observational study. The primary outcome was the duration of symptoms rated moderately bad or worse. Throat swabs were taken and analyzed for the presence of bacteria and viruses by multiplex PCR. Results Swab results were available for most participants in the trial (306 of 432; 71%) and in the observational (182 of 326; 59%) studies. Bacterial pathogens potentially sensitive to amoxicillin (Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae) were detected among 51% of the trial placebo group and 49% of the trial antibiotic group. The median difference in the duration of symptoms rated moderately bad or worse between antibiotic and placebo was similar when potentially antibiotic-susceptible bacteria were present (median: –1 day; 99% CI, –12.3 to 10.3) or not present (median: –1 day; 99% CI, –4.5 to 2.5). Furthermore, bacterial genome copy number did not predict benefit. There were similar findings for all secondary outcomes and when including the data from the observational study. Discussion There was no clear evidence that antibiotics improved clinical outcomes conditional on the presence or concentration of bacteria or viruses in the upper airway. Before deploying microbiologic point-of-care tests for children with uncomplicated LRTI in primary care, rigorous validating trials are needed

Comparison of microbiological diagnosis of urinary tract infection in young children by routine health service laboratories and a research laboratory: Diagnostic cohort study

OBJECTIVES: To compare the validity of diagnosis of urinary tract infection (UTI) through urine culture between samples processed in routine health service laboratories and those processed in a research laboratory. POPULATION AND METHODS: We conducted a prospective diagnostic cohort study in 4808 acutely ill children aged <5 years attending UK primary health care. UTI, defined as pure/predominant growth ≥105 CFU/mL of a uropathogen (the reference standard), was diagnosed at routine health service laboratories and a central research laboratory by culture of urine samples. We calculated areas under the receiver-operator curve (AUC) for UTI predicted by pre-specified symptoms, signs and dipstick test results (the "index test"), separately according to whether samples were obtained by clean catch or nappy (diaper) pads. RESULTS: 251 (5.2%) and 88 (1.8%) children were classified as UTI positive by health service and research laboratories respectively. Agreement between laboratories was moderate (kappa = 0.36; 95% confidence interval [CI] 0.29, 0.43), and better for clean catch (0.54; 0.45, 0.63) than nappy pad samples (0.20; 0.12, 0.28). In clean catch samples, the AUC was lower for health service laboratories (AUC = 0.75; 95% CI 0.69, 0.80) than the research laboratory (0.86; 0.79, 0.92). Values of AUC were lower in nappy pad samples (0.65 [0.61, 0.70] and 0.79 [0.70, 0.88] for health service and research laboratory positivity, respectively) than clean catch samples. CONCLUSIONS: The agreement of microbiological diagnosis of UTI comparing routine health service laboratories with a research laboratory was moderate for clean catch samples and poor for nappy pad samples and reliability is lower for nappy pad than for clean catch samples. Positive results from the research laboratory appear more likely to reflect real UTIs than those from routine health service laboratories, many of which (particularly from nappy pad samples) could be due to contamination. Health service laboratories should consider adopting procedures used in the research laboratory for paediatric urine samples. Primary care clinicians should try to obtain clean catch samples, even in very young children