Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

Comparative accuracy and cost-effectiveness of dynamic contrast-enhanced CT and positron emission tomography in the characterisation of solitary pulmonary nodules.

INTRODUCTION: Dynamic contrast-enhanced CT (DCE-CT) and positron emission tomography/CT (PET/CT) have a high reported accuracy for the diagnosis of malignancy in solitary pulmonary nodules (SPNs). The aim of this study was to compare the accuracy and cost-effectiveness of these. METHODS: In this prospective multicentre trial, 380 participants with an SPN (8-30 mm) and no recent history of malignancy underwent DCE-CT and PET/CT. All patients underwent either biopsy with histological diagnosis or completed CT follow-up. Primary outcome measures were sensitivity, specificity and overall diagnostic accuracy for PET/CT and DCE-CT. Costs and cost-effectiveness were estimated from a healthcare provider perspective using a decision-model. RESULTS: 312 participants (47% female, 68.1±9.0 years) completed the study, with 61% rate of malignancy at 2 years. The sensitivity, specificity, positive predictive value and negative predictive values for DCE-CT were 95.3% (95% CI 91.3 to 97.5), 29.8% (95% CI 22.3 to 38.4), 68.2% (95% CI 62.4% to 73.5%) and 80.0% (95% CI 66.2 to 89.1), respectively, and for PET/CT were 79.1% (95% CI 72.7 to 84.2), 81.8% (95% CI 74.0 to 87.7), 87.3% (95% CI 81.5 to 91.5) and 71.2% (95% CI 63.2 to 78.1). The area under the receiver operator characteristic curve (AUROC) for DCE-CT and PET/CT was 0.62 (95% CI 0.58 to 0.67) and 0.80 (95% CI 0.76 to 0.85), respectively (p<0.001). Combined results significantly increased diagnostic accuracy over PET/CT alone (AUROC=0.90 (95% CI 0.86 to 0.93), p<0.001). DCE-CT was preferred when the willingness to pay per incremental cost per correctly treated malignancy was below £9000. Above £15 500 a combined approach was preferred. CONCLUSIONS: PET/CT has a superior diagnostic accuracy to DCE-CT for the diagnosis of SPNs. Combining both techniques improves the diagnostic accuracy over either test alone and could be cost-effective. TRIAL REGISTRATION NUMBER: NCT02013063.

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels.

The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.

Temporal characterization of protein production levels from baculovirus vectors coding for GFP and RFP genes under non-conventional promoter control.

The ease of use and versatility of the Baculovirus Expression Vector System (BEVS) has made it one of the most widely used systems for recombinant protein production However, co-expression systems currently in use mainly make use of the very strong very late p10 and polyhedron (polh) promoters to drive expression of foreign genes, which does not provide much scope for tailoring expression ratios within the cell. This work demonstrates the use of different Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) promoters to control the timing and expression of two easily traceable fluorescent proteins, the enhanced green fluorescent protein (eGFP), and a red fluorescent protein (DsRed2) in a BEVS co-expression system. Our results show that gene expression levels can easily be controlled using this strategy, and also that modulating the expression level of one protein can influence the level of expression of the other protein within the system, thus confirming the concept of genes "competing" for limited cellular resources. Plots of "expression ratios" of the two model genes over time were obtained, and may be used in future work to tightly control timing and levels of foreign gene expression in an insect cell co-expression system.

A systematic review of symptomatic diagnosis of lung cancer.

BACKGROUND: Lung cancer (LC) is often diagnosed late when curative intervention is no longer viable. However, current referral guidelines (e.g. UK National Institute for Health and Care Excellence guidelines) for suspected LC are based on a weak evidence base. Aim. The purpose of this systematic review is to identify symptoms that are independently associated with LC and to identify the key methodological issues relating to symptomatic diagnosis research in LC. METHODS: Medline, Ovid and Cumulative Index to Nursing and Allied Health Literature were searched for the period between 1946 and 2012 using the MeSH terms 'lung cancer' and 'symptom*'. Quality of each paper was assessed using Scottish Intercollegiate Guidelines Network and Consolidated Criteria for Reporting Qualitative Research Checklists and checked by a second and third reviewer. RESULTS: Evidence regarding the diagnostic values of most symptoms was inconclusive; haemoptysis was the only symptom consistently indicated as a predictor of LC. Generally, evidence was weakened by methodological issues such as the lack of standardized data collection (recording bias) and the lack of comparability of findings across the different studies that extend beyond the spectrum of disease. Qualitative studies indicated that patients with LC experienced symptoms months before diagnosis but did not interpret them as serious enough to seek health care. Therefore, early LC symptoms might be under-represented in primary care clinical notes. CONCLUSION: Current evidence is insufficient to suggest a symptom profile for LC across the disease stages, nor can it be concluded that classical LC symptoms are predictors of LC apart from, perhaps, haemoptysis. Prospective studies are now needed that systematically record symptoms and explore their predictive values for LC diagnosis.

Effect of 3 to 5 years of scheduled CEA and CT follow-up to detect recurrence of colorectal cancer: the FACS randomized clinical trial.

IMPORTANCE: Intensive follow-up after surgery for colorectal cancer is common practice but is based on limited evidence. OBJECTIVE: To assess the effect of scheduled blood measurement of carcinoembryonic antigen (CEA) and computed tomography (CT) as follow-up to detect recurrent colorectal cancer treatable with curative intent. DESIGN, SETTING, AND PARTICIPANTS: Randomized clinical trial in 39 National Health Service hospitals in the United Kingdom; 1202 eligible participants were recruited between January 2003 and August 2009 who had undergone curative surgery for primary colorectal cancer, including adjuvant treatment if indicated, with no evidence of residual disease on investigation. INTERVENTIONS: Participants were randomly assigned to 1 of 4 groups: CEA only (n = 300), CT only (n = 299), CEA+CT (n = 302), or minimum follow-up (n = 301). Blood CEA was measured every 3 months for 2 years, then every 6 months for 3 years; CT scans of the chest, abdomen, and pelvis were performed every 6 months for 2 years, then annually for 3 years; and the minimum follow-up group received follow-up if symptoms occurred. MAIN OUTCOMES AND MEASURES: The primary outcome was surgical treatment of recurrence with curative intent; secondary outcomes were mortality (total and colorectal cancer), time to detection of recurrence, and survival after treatment of recurrence with curative intent. RESULTS: After a mean 4.4 (SD, 0.8) years of observation, cancer recurrence was detected in 199 participants (16.6%; 95% CI, 14.5%-18.7%) overall; 71 of 1202 participants (5.9%; 95% CI, 4.6%-7.2%) were treated for recurrence with curative intent, with little difference according to Dukes staging (stage A, 5.1% [13/254]; stage B, 6.1% [34/553]; stage C, 6.2% [22/354]). Surgical treatment of recurrence with curative intent was 2.3% (7/301) in the minimum follow-up group, 6.7% (20/300) in the CEA group, 8% (24/299) in the CT group, and 6.6% (20/302) in the CEA+CT group. Compared with minimum follow-up, the absolute difference in the percentage of patients treated with curative intent in the CEA group was 4.4% (95% CI, 1.0%-7.9%; adjusted odds ratio [OR], 3.00; 95% CI, 1.23-7.33), in the CT group was 5.7% (95% CI, 2.2%-9.5%; adjusted OR, 3.63; 95% CI, 1.51-8.69), and in the CEA+CT group was 4.3% (95% CI, 1.0%-7.9%; adjusted OR, 3.10; 95% CI, 1.10-8.71). The number of deaths was not significantly different in the combined intensive monitoring groups (CEA, CT, and CEA+CT; 18.2% [164/901]) vs the minimum follow-up group (15.9% [48/301]; difference, 2.3%; 95% CI, -2.6% to 7.1%). CONCLUSIONS AND RELEVANCE: Among patients who had undergone curative surgery for primary colorectal cancer, intensive imaging or CEA screening each provided an increased rate of surgical treatment of recurrence with curative intent compared with minimal follow-up; there was no advantage in combining CEA and CT. If there is a survival advantage to any strategy, it is likely to be small. TRIAL REGISTRATION: isrctn.org Identifier: 41458548.

Percutaneous cryoablation of renal tumours: outcomes from 171 tumours in 147 patients.

OBJECTIVE: To evaluate the technical and oncological efficacy of an image-guided cryoablation programme for renal tumours. PATIENTS AND METHODS: A prospective analysis of technical and radiological outcomes was undertaken after treatment of 171 consecutive tumours in 147 patients. Oncological efficacy in a subset of 125 tumours in 104 patients with >6 months' radiological follow-up and a further subset of 62 patients with solitary, biopsy-proven renal carcinoma was also analysed. Factors influencing technical success, as determined by imaging follow-up, and complication rates were statistically analysed using a statistics software package and logistic regression analyses. RESULTS: No variables were found to predict subtotal treatment, although gender (P = 0.08), tumour size of >4 cm (P = 0.09) and central location of tumour (P = 0.07) approached significance. Upper pole location was the single variable that was found to predict complications (P = 0.006). Among the 104 patients (125 tumours), radiologically assessed at ≥6 months and with a mean radiological follow-up of 20.1 months, we found a single case of unexpected late local recurrence. CONCLUSION: Percutaneous image-guided cryoablation, at a mean of 20.1 months' follow-up, appears to provide a safe and effective treatment option with a low complication rate. Anteriorly sited tumours should not be considered a contraindication for percutaneous image-guided cryoablation.

Dynamic contrast-enhanced CT compared with positron emission tomography CT to characterise solitary pulmonary nodules: the SPUtNIk diagnostic accuracy study and economic modelling.

BACKGROUND: Current pathways recommend positron emission tomography-computerised tomography for the characterisation of solitary pulmonary nodules. Dynamic contrast-enhanced computerised tomography may be a more cost-effective approach. OBJECTIVES: To determine the diagnostic performances of dynamic contrast-enhanced computerised tomography and positron emission tomography-computerised tomography in the NHS for solitary pulmonary nodules. Systematic reviews and a health economic evaluation contributed to the decision-analytic modelling to assess the likely costs and health outcomes resulting from incorporation of dynamic contrast-enhanced computerised tomography into management strategies. DESIGN: Multicentre comparative accuracy trial. SETTING: Secondary or tertiary outpatient settings at 16 hospitals in the UK. PARTICIPANTS: Participants with solitary pulmonary nodules of ≥ 8 mm and of ≤ 30 mm in size with no malignancy in the previous 2 years were included. INTERVENTIONS: Baseline positron emission tomography-computerised tomography and dynamic contrast-enhanced computer tomography with 2 years' follow-up. MAIN OUTCOME MEASURES: Primary outcome measures were sensitivity, specificity and diagnostic accuracy for positron emission tomography-computerised tomography and dynamic contrast-enhanced computerised tomography. Incremental cost-effectiveness ratios compared management strategies that used dynamic contrast-enhanced computerised tomography with management strategies that did not use dynamic contrast-enhanced computerised tomography. RESULTS: A total of 380 patients were recruited (median age 69 years). Of 312 patients with matched dynamic contrast-enhanced computer tomography and positron emission tomography-computerised tomography examinations, 191 (61%) were cancer patients. The sensitivity, specificity and diagnostic accuracy for positron emission tomography-computerised tomography and dynamic contrast-enhanced computer tomography were 72.8% (95% confidence interval 66.1% to 78.6%), 81.8% (95% confidence interval 74.0% to 87.7%), 76.3% (95% confidence interval 71.3% to 80.7%) and 95.3% (95% confidence interval 91.3% to 97.5%), 29.8% (95% confidence interval 22.3% to 38.4%) and 69.9% (95% confidence interval 64.6% to 74.7%), respectively. Exploratory modelling showed that maximum standardised uptake values had the best diagnostic accuracy, with an area under the curve of 0.87, which increased to 0.90 if combined with dynamic contrast-enhanced computerised tomography peak enhancement. The economic analysis showed that, over 24 months, dynamic contrast-enhanced computerised tomography was less costly (£3305, 95% confidence interval £2952 to £3746) than positron emission tomography-computerised tomography (£4013, 95% confidence interval £3673 to £4498) or a strategy combining the two tests (£4058, 95% confidence interval £3702 to £4547). Positron emission tomography-computerised tomography led to more patients with malignant nodules being correctly managed, 0.44 on average (95% confidence interval 0.39 to 0.49), compared with 0.40 (95% confidence interval 0.35 to 0.45); using both tests further increased this (0.47, 95% confidence interval 0.42 to 0.51). LIMITATIONS: The high prevalence of malignancy in nodules observed in this trial, compared with that observed in nodules identified within screening programmes, limits the generalisation of the current results to nodules identified by screening. CONCLUSIONS: Findings from this research indicate that positron emission tomography-computerised tomography is more accurate than dynamic contrast-enhanced computerised tomography for the characterisation of solitary pulmonary nodules. A combination of maximum standardised uptake value and peak enhancement had the highest accuracy with a small increase in costs. Findings from this research also indicate that a combined positron emission tomography-dynamic contrast-enhanced computerised tomography approach with a slightly higher willingness to pay to avoid missing small cancers or to avoid a 'watch and wait' policy may be an approach to consider. FUTURE WORK: Integration of the dynamic contrast-enhanced component into the positron emission tomography-computerised tomography examination and the feasibility of dynamic contrast-enhanced computerised tomography at lung screening for the characterisation of solitary pulmonary nodules should be explored, together with a lower radiation dose protocol. STUDY REGISTRATION: This study is registered as PROSPERO CRD42018112215 and CRD42019124299, and the trial is registered as ISRCTN30784948 and ClinicalTrials.gov NCT02013063. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 26, No. 17. See the NIHR Journals Library website for further project information.

A nodule found on a lung scan can cause concern as it may be a sign of cancer. Finding lung cancer nodules when they are small (i.e. < 3 cm) is very important. Most nodules are not cancerous. Computerised tomography (cross-sectional images created from multiple X-rays) and positron emission tomography­computerised tomography (a technique that uses a radioactive tracer combined with computerised tomography) are used to see whether or not a nodule is cancerous; although they perform well, improvements are required. This study compared dynamic contrast-enhanced computerised tomography with positron emission tomography­computerised tomography scans to find out which test is best. Dynamic contrast-enhanced computerised tomography involves injection of a special dye into the bloodstream, followed by repeated scans of the nodule over several minutes. We assessed the costs to the NHS of undertaking the different scans, relative to their benefits, to judge which option was the best value for money. We recruited 380 patients from 16 hospitals across England and Scotland, of whom 312 had both dynamic contrast-enhanced computerised tomography and positron emission tomography­computerised tomography scans. We found that current positron emission tomography­computerised tomography is more accurate, providing a correct diagnosis in 76% of cases, than the new dynamic contrast-enhanced computerised tomography, which provides a correct diagnosis in 70% of cases. Although dynamic contrast-enhanced computerised tomography cannot replace positron emission tomography­computerised tomography, it may represent good-value use of NHS resources, especially if it is performed before positron emission tomography­computerised tomography and they are used in combination. Although more research is required, it may be possible in the future to perform dynamic contrast-enhanced computerised tomography at the same time as positron emission tomography­computerised tomography in patients with suspected lung cancer or if a lung nodule is found on a lung screening programme at the time of the computerised tomography examination. This may reduce the need for some people to have positron emission tomography­computerised tomography.

Isolating and characterizing lymphatic endothelial progenitor cells for potential therapeutic lymphangiogenic applications.

Lymphatic dysfunction is associated with the progression of several vascular disorders, though currently, there are limited strategies to promote new lymphatic vasculature (i.e., lymphangiogenesis) to restore lost lymphatic function. One promising approach to stimulate lymphangiogenesis involves delivering endothelial progenitor cells (EPCs), which are naturally involved in de novo blood vessel formation and have recently been identified to include a lymphatic subpopulation. However, the contribution of lymphatic EPCs in lymphangiogenesis is not clear and challenges with maintaining the activity of transplanted EPCs remain. Thus, the objective of this study was to isolate lymphatic EPCs from human umbilical cord blood and characterize their role in the initial stages of blood or lymphatic vasculature formation. Furthermore, this study also tested the applicability of alginate hydrogels to deliver lymphatic EPCs for a possible therapeutic application. We postulated and confirmed that blood and lymphatic EPC colonies could be isolated from human umbilical cord blood. Additionally, EPC populations responded to either angiogenic or lymphangiogenic growth factors and could stimulate their respective mature endothelial cells in vasculature models in vitro. Finally, lymphatic EPCs maintained their ability to promote lymphatic sprouts after prolonged interactions with the alginate hydrogel microenvironment. These results suggest EPCs have both a blood and a lymphatic population that have specific roles in promoting revascularization and highlight the potential of alginate hydrogels for the delivery of lymphatic EPCs. STATEMENT OF SIGNIFICANCE: Despite the potential therapeutic benefit of promoting lymphatic vasculature, lymphangiogenesis remains understudied. One appealing strategy for promoting lymphangiogenesis involves delivering lymphatic endothelial progenitor cells (EPCs), which are a subpopulation of EPCs involved in de novo vessel formation. Here, we investigate the role of isolated blood and lymphatic EPC subpopulations in promoting the early stages of vascularization and the utility of alginate hydrogels to deliver lymphatic EPCs. We determined that EPCs had two populations that expressed either blood or lymphatic markers, could stimulate their respective mature vasculature in tissue constructs and that alginate hydrogels maintained the therapeutic potential of lymphatic EPCs. We anticipate this work could support promising biomaterial applications of EPCs to promote revascularization, which could have many therapeutic applications.

The subjective index for physical and social outcome (SIPSO) in stroke: investigation of its subscale structure.

BACKGROUND: Short and valid measures of the impact of a stroke on integration are required in health and social settings. The Subjective Index of Physical and Social Outcome (SIPSO) is one such measure. However, there are questions whether scores can be summed into a total score or whether subscale scores should be calculated. This paper aims to provide clarity on the internal construct validity of the subscales and the total scale. METHODS: SIPSO data were collected as part of two parallel surveys of the met and unmet needs of 445 younger people (aged 18-65) with non-recent stroke (at least one year) and living at home. Factor, Mokken and Rasch analysis were used. RESULTS: Factor analysis supported a two factor structure (explaining 68% of the variance) as did the Mokken analysis (overall Loevinger coefficient 0.77 for the Physical Integration subscale; 0.51 for the Social Integration subscale). Both subscales fitted the Rasch model (P > 0.01) after adjusting for some observed differential item functioning. The 10-items together did not fit the Rasch model. CONCLUSIONS: The SIPSO subscales are valid for use with stroke patients of working age but the total SIPSO is not. The conversion table can be used by clinicians and researchers to convert ordinal data to interval level prior to mathematical operations and other parametric procedures. Further work is required to explore the occurrence of bias by gender for some of the items.

The Winchester falls project: a randomised controlled trial of secondary prevention of falls in older people.

BACKGROUND: the mortality and morbidity of falls in older people is significant, with recurrent fallers being at an increased risk. The most effective way to reduce falls in this group is not clear. OBJECTIVE: to determine the effectiveness of two interventions, one based in primary care and the other in secondary care, at preventing further falls in recurrent fallers. DESIGN: cluster randomised controlled trial. PARTICIPANTS: sixty-five years or over, living in the community, two or more falls in the previous year and not presenting to an emergency department with index fall. SETTING: Mid Hampshire, UK. INTERVENTION: eighteen general practices were randomly allocated to one of three groups. The primary care group was assessed by nurses in the community, using a risk factor review and subsequent targeted referral to other professionals. The secondary care group received a multi-disciplinary assessment in a day hospital followed by identified appropriate interventions. The control group received usual care. Follow-up was for 1 year. RESULTS: five hundred and five participants were recruited. Follow-up was completed in 83% (421/505). The proportion of participants who fell again was significantly lower in the secondary care group (75%, 158/210) compared to the control group [84%, 133/159, adjusted odds ratio (OR) 0.52 (95% CI 0.35-0.79) P = 0.002]. The primary care group showed similar results to the control group [87%, 118/136, adjusted OR 1.17 (95% CI 0.57-2.37) P = 0.673]. CONCLUSION: a structured multi-disciplinary assessment of recurrent fallers significantly reduced the number experiencing further falls, but a community-based nurse-led assessment with targeted referral to other professionals did not.

A flow cytometric granularity assay for the quantification of infectious virus.

A flow cytometry-based assay was developed to assess the infective titer of two recombinant viruses: a recombinant herpes simplex type 2 (rHSV-2) and a recombinant canary pox (rALVAC.gfp). This method uses granularity of infected Vero and QT-35 cells, respectively, and correlates this to the infectious titer of virus samples. The percent of the cell populations with a high level of granularity could accurately be correlated to viral titers obtained through a traditional plaque assay, with R2 values greater than 0.8 using a semi-logarithmic scale. This approach offers a rapid, high-throughput method for infectious virus titration with similar accuracy to a traditional plaque assay.

Dual-function growth medium and structural soil for use as porous pavement.

Permeable grass-covered surfaces can reduce the quantity of storm water runoff and filter out potentially harmful chemicals. The objective of this study was to develop permeable structural soils that sustained healthy turf growth and filtered heavy metals from contaminated pavement runoff. The basic soil medium was a 50:50 mixture (v/v) of expanded shale (ExSh) and quartz sand (QS). The ExSh component consisted of (i) large-diameter particles (3-6 mm), (ii) small-diameter particles (1-3 mm), or (iii) a 50:50 mixture (v/v) of the two. The basic blends were mixed with 0, 10, and 20% sphagnum peat moss (v/v) and 0, 10, and 20% natural zeolites (v/v) and placed in 15-cm-diameter pots in a greenhouse. Bermudagrass plugs were planted in each pot. The addition of sphagnum peat moss to the basic ExSh/QS blend increased bermudagrass growth and improved plant response to added fertilizer. Zeolites had no significant effect on plant growth in the absence of sphagnum peat moss. Growing mediums that contained 10 to 20% sphagnum peat moss and 10 to 20% zeolites consistently produced more bermudagrass biomass than the unamended ExSh/QS mixture. Changing the ratio of small- to large-diameter ExSh in the basic medium did not affect bermudagrass yield. Very low amounts of Cd, Cu, Pb, and Zn were recovered in leachate after the addition of 10 mg metal per pot, suggesting that most heavy metals (>99%) were retained in the growing mediums. Zeolites reduced the amount of Cd and Pb in leachate water, but not Cu or Zn.

The analytical challenges of cyclopropylfentanyl and crotonylfentanyl: An approach for toxicological analysis.

New psychoactive substances (NPS) are increasingly being seen in forensic casework globally and encompass a number of types of drugs including "designer opioids", especially fentanyl analogues, which are of particular concern due to their high potency and significant risk of toxicity. They are often sold as heroin or mixed with other illicit drugs and therefore users may be unaware they are taking such hazardous compounds. Two fentanyl analogues that have recently been detected are cyclopropylfentanyl and crotonylfentanyl. In order to accurately determine the prevalence of such compounds in clinical and forensic casework, including potential toxicity, they need to be correctly identified using definitive and defensible techniques. Cyclopropylfentanyl and crotonylfentanyl are structural isomers, and it has previously been highlighted that these 2 compounds are analytically difficult to specifically identify owing to their similarity in structure and chromatographic behaviour. To further investigate in an attempt to overcome this problem, analysis of certified reference material using high performance liquid chromatography with diode array UV detection (HPLC-DAD), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QToF-MS) has been performed. Whilst the compounds were shown to have an identical mass-spectral fragmentation pattern, they had different UV spectra. This was coupled with a discernible difference in retention time with the HPLC conditions applied, allowing differentiation of the 2 compounds. Using this approach, cyclopropylfentanyl was positively identified and subsequently quantified in 4 fatalities with the exclusion of crotonylfentanyl.

Impact of rising sea levels on Australian fur seals.

Global warming is leading to many unprecedented changes in the ocean-climate system. Sea levels are rising at an increasing rate and are amplifying the impact of storm surges along coastlines. As variability in the timing and strength of storm surges has been shown to affect pup mortality in the Australian fur seal (Arctocephalus pusillus doriferus), there is a need to identify the potential impacts of increased sea level and storm surges on the breeding areas of this important marine predator in Bass Strait, south-eastern Australia. Using high-resolution aerial photography and topographic data, the present study assessed the impacts of future inundation levels on both current and potential breeding habitats at each colony. Inundation from storm surges, based on a predicted rise in sea level, was modeled at each colony from 2012 to 2100. As sea level increases, progressively less severe storm surge conditions will be required to exceed current inundation levels and, thus, have the potential for greater impacts on pup mortality at Australian fur seal colonies. The results of the present study indicate that by 2100, a 1-in-10 year storm will inundate more habitat on average than a present-day 1-in-100 year storm. The study highlights the site-specific nature of storm surge impacts, and in particular the importance of local colony topography and surrounding bathymetry with small, low-lying colonies impacted the most. An increased severity of storm surges will result in either an increase in pup mortality rates associated with storm surges, or the dispersal of individuals to higher ground and/or new colonies.

Physiotherapy breathing retraining for asthma: a randomised controlled trial.

BACKGROUND: Despite effective pharmacotherapy, asthma continues to impair quality of life for most patients. Non-pharmacological approaches, including breathing retraining, are therefore of great interest to patients. However, clinicians rarely advocate breathing retraining and access to this intervention is restricted for most patients due to the limited availability of suitable physiotherapists and poor integration of breathing retraining into standard care. We aimed to assess the effectiveness of a digital self-guided breathing retraining intervention. METHODS: In this randomised controlled trial, we recruited patients from 34 general practices in the UK. Eligibility criteria for patients with asthma were broad, comprising a physician diagnosis of asthma, age of 16-70 years, receipt of at least one anti-asthma medication in the previous year, and impaired asthma-related quality of life (Asthma Quality of Life Questionnaire [AQLQ] score of <5·5). We developed a self-guided intervention, which was delivered as a DVD plus a printed booklet (DVDB). Participants were randomly assigned to receive either the DVDB intervention, three face-to-face breathing retraining sessions, or standard care, in a 2:1:2 ratio, for 12 months. Randomisation was achieved using the Southampton Clinical Trials Unit telephone randomisation service by use of random number generators. The primary outcome was the AQLQ score in the intention-to-treat population at 12 months. The trial was powered to show equivalence between the two active intervention groups, and superiority of both intervention groups over usual care. Secondary outcomes included patient-reported and physiological measures of asthma control, patient acceptability, and health-care costs. This trial was registered with International Standard Randomised Controlled Trial Number registry, number ISRCTN88318003. FINDINGS: Between Nov 5, 2012 and Jan 28, 2014, invitations to participate in the study were sent to 15 203 patients with general practitioner-diagnosed asthma, of whom 655 were recruited into the study. AQLQ scores at 12 months were significantly higher in the DVDB group (mean 5·40, SD 1·14) than in the usual care group (5·12, SD 1·17; adjusted mean difference 0·28, 95% CI 0·11 to 0·44), and in the face-to-face group (5·33, SD 1·06) than in the usual care group (adjusted mean difference 0·24, 95% CI 0·04 to 0·44); AQLQ scores were similar between the DVDB group and the face-to-face group (0·04, 95% CI -0·16 to 0·24). There were no significant differences between the randomisation groups in FEV1 or fraction of exhaled nitric oxide. 744 adverse events occurred in 272 patients: 101 (39%) of 261 patients in the DVDB group, 55 (42%) of 132 patients in the face-to-face group, and 132 (50%) of 262 in the usual care group, with patients reporting one or more event. 11 (4%) patients in the DVDB group, four (3%) patients in the face-to-face group, and 20 (8%) patients in the usual care group had a serious adverse event. INTERPRETATION: Breathing retraining programmes improve quality of life in patients with incompletely controlled asthma despite having little effect on lung function or airway inflammation. Such programmes can be delivered conveniently and cost-effectively as a self-guided digital audiovisual programme, so might also reduce health-care costs. FUNDING: UK National Institute of Health Research.

Validity of the impact on participation and autonomy questionnaire: a comparison between two countries.

PURPOSE: To evaluate the cross-cultural validity of the five subscales of the Impact on Participation and Autonomy (IPA) measure and the full 31-item scale. METHOD: Data from two validation studies (Dutch and English) were pooled (n = 106). Participants (aged 18-75), known to rehabilitation services or GP practices, had conditions ranging from minor ailments to significant disability. Validity of the five subscales and the total scale was examined using Rasch analysis (Partial Credit Model). P values smaller than 0.01 were employed to allow for multiple testing. RESULTS: A number of items in all the subscales except 'Outdoor Autonomy' needed rescoring. One 'Indoor Autonomy' item showed uniform DIF by country and was split by country. One 'Work and Education' item displayed uniform and non-uniform DIF by gender. All the subscales fitted the Rasch model and were invariant across country. A 30-item IPA also fitted the Rasch model. CONCLUSION: The IPA subscales and a 30-item scale are invariant across the two cultures and gender. The IPA can be used validly to assess participation and autonomy in these populations. Further analyses are required to examine whether the IPA is invariant across differing levels of disability and other disease groups not included in this study.

A randomised controlled study of the effectiveness of breathing retraining exercises taught by a physiotherapist either by instructional DVD or in face-to-face sessions in the management of asthma in adults.

BACKGROUND: Asthma control is suboptimal, resulting in quality of life (QoL) impairment and costs. Breathing retraining exercises have evidence of effectiveness as adjuvant treatment, but are infrequently used. OBJECTIVES: To transfer the contents of a brief (three-session) physiotherapist-delivered breathing retraining programme to a digital versatile disc (DVD) and booklet format; to compare the effectiveness of the self-guided intervention with that of 'face-to-face' physiotherapy and usual care for QoL and other asthma-related outcomes; to perform a health economic assessment of both interventions; and to perform a process evaluation using quantitative and qualitative methods. DESIGN: Parallel-group three-arm randomised controlled trial. SETTING: General practice surgeries in the UK. PARTICIPANTS: In total, 655 adults currently receiving asthma treatment with impaired asthma-related QoL were randomly allocated to the DVD (n = 261), physiotherapist (n = 132) and control (usual care) (n = 262) arms in a 2 : 1 : 2 ratio. It was not possible to blind participants but data collection and analysis were performed blinded. INTERVENTIONS: Physiotherapy-based breathing retraining delivered through three 'face-to-face' respiratory physiotherapist sessions or a self-guided programme (DVD plus our theory-based behaviour change booklet) developed by the research team, with a control of usual care. MAIN OUTCOME MEASURES: The primary outcome measure was asthma-specific QoL, measured using the Asthma Quality of Life Questionnaire (AQLQ). Secondary outcomes included asthma symptom control [Asthma Control Questionnaire (ACQ)], psychological state [Hospital Anxiety and Depression Scale (HADS)], hyperventilation symptoms (Nijmegen questionnaire), generic QoL [EuroQol-5 Dimensions (EQ-5D)], assessments of airway physiology (spirometry) and inflammation (exhaled nitric oxide) and health resource use and costs. Assessments were carried out at baseline and at 3, 6 and 12 months post randomisation. Patient engagement and experience were also assessed using quantitative and qualitative methods. RESULTS: Primary efficacy analysis was between-group comparison of changes in AQLQ scores from baseline to 12 months in the intention-to-treat population with adjustments for prespecified covariates. Significant improvements occurred in the DVD group compared with the control group [adjusted mean difference 0.28, 95% confidence interval (CI) 0.11 to 0.44; p < 0.001] and in the face-to-face physiotherapy group compared with the control group (adjusted mean difference 0.24, 95% CI 0.04 to 0.44; p < 0.05), with equivalence between the DVD and the face-to-face physiotherapy groups (adjusted mean difference 0.04, 95% CI -0.16 to 0.24). In all sensitivity analyses, both interventions remained significantly superior to the control and equivalence between the interventions was maintained. In other questionnaire outcome measures and in the physiological measures assessed, there were no significant between-group differences. Process evaluations showed that participants engaged well with both of the active interventions, but that some participants in the DVD arm would have liked to receive tuition from a professional. Asthma health-care costs were lower in both intervention arms than in the control group, indicating 'dominance' for both of the interventions compared with the control, with lowest costs in the DVD arm. The rate of adverse events was lower in the DVD and face-to-face physiotherapy groups than in the control group. CONCLUSIONS: Only 10% of the potentially eligible population responded to the study invitation. However, breathing retraining exercises improved QoL and reduced health-care costs in adults with asthma whose condition remains uncontrolled despite standard pharmacological therapy, were engaged with well by patients and can be delivered effectively as a self-guided intervention. The intervention should now be transferred to an internet-based platform and implementation studies performed. Interventions for younger patients should be developed and trialled. TRIAL REGISTRATION: Current Controlled Trials ISRCTN88318003. FUNDING: This project was primarily funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 53. See the NIHR Journals Library website for further project information. Additional financial support was received from Comprehensive Local Research Networks.

A systematic review of evidence about extended roles for allied health professionals.

OBJECTIVE: Extending the role of allied health professionals has been promoted as a key component of developing a flexible health workforce. This review aimed to synthesize the evidence about the impact of these roles. METHODS: A systematic review of extended scope of practice in five groups: paramedics, physiotherapists, occupational therapists, radiographers, and speech and language therapists. The nature and effect of these roles on patients, health professionals and health services were examined. An inclusive approach to searching was used to maximize potential sources of interest including multiple databases, 'grey' literature and subject area experts. An expanded Cochrane Collaboration method was used in view of the anticipated lack of randomized controlled trials and heterogeneity of designs. Papers were only excluded after the search stage for lack of relevance. RESULTS: A total of 355 papers was identified as meeting relevance criteria and 21 studies progressed to full review and data extraction. The primary reason for exclusion from data extraction was that the study included neither qualitative nor quantitative data or because methodological flaws compromised data quality. It was not possible to evaluate any pooled effects as patient health outcomes were rarely considered. CONCLUSIONS: A range of extended practice roles for allied health professionals have been promoted and are being undertaken, but their health outcomes have rarely been evaluated. There is also little evidence as to how best to introduce such roles, or how best to educate, support and mentor these practitioners.