Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE.

The steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different subcellular niches upon perturbation of the subcellular environment. Differential localisation, that is a change in the steady-state subcellular location of a protein, provides a step towards mechanistic insight of subcellular protein dynamics. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we describe a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to several datasets recovers well-studied translocations. In an application to cytomegalovirus infection, we obtain insights into the rewiring of the host proteome. Integration of other high-throughput datasets allows us to provide the functional context of these data.

Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation.

Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells.

Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist.

Vaccinia virus (VACV) has numerous immune evasion strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor κB (NF-κB), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics to quantify ∼9,000 cellular proteins and ∼80% of viral proteins at seven time points throughout VACV infection. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during infection. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a protein-based temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of HDAC5. Our approach thus identifies both a host antiviral factor and a viral mechanism of innate immune evasion.

Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G2 Arrest and Highly Effective Evasion of Innate Immune Sensing.

BK polyomavirus (BKPyV) is a small DNA virus that establishes a life-long persistent infection in the urinary tract of most people. BKPyV is known to cause severe morbidity in renal transplant recipients and can lead to graft rejection. The simple 5.2-kbp double-stranded DNA (dsDNA) genome expresses just seven known proteins; thus, it relies heavily on the host machinery to replicate. How the host proteome changes over the course of infection is key to understanding this host-virus interplay. Here, for the first time quantitative temporal viromics has been used to quantify global changes in >9,000 host proteins in two types of primary human epithelial cells throughout 72 h of BKPyV infection. These data demonstrate the importance of cell cycle progression and pseudo-G2 arrest in effective BKPyV replication, along with a surprising lack of an innate immune response throughout the whole virus replication cycle. BKPyV thus evades pathogen recognition to prevent activation of innate immune responses in a sophisticated manner.IMPORTANCE BK polyomavirus can cause serious problems in immune-suppressed patients, in particular, kidney transplant recipients who can develop polyomavirus-associated kidney disease. In this work, we have used advanced proteomics techniques to determine the changes to protein expression caused by infection of two independent primary cell types of the human urinary tract (kidney and bladder) throughout the replication cycle of this virus. Our findings have uncovered new details of a specific form of cell cycle arrest caused by this virus, and, importantly, we have identified that this virus has a remarkable ability to evade detection by host cell defense systems. In addition, our data provide an important resource for the future study of kidney epithelial cells and their infection by urinary tract pathogens.

Cell Cycle Arrest is a Conserved Function of Norovirus VPg Proteins.

Murine norovirus (MNV) viral protein genome-linked (VPg) manipulates the cell cycle to induce a G0/G1 arrest and gain a beneficial replication environment. All viruses of the norovirus genus encode a VPg protein; however, it is unknown if the G0/G1 arrest induced by MNV VPg is conserved in other members of the genus. RNA transcripts encoding a representative viral VPg from five norovirus genogroups were transfected into RAW-Blue murine macrophages, and the percentage of cells in each phase of the cell cycle was determined. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins tested, and in the wider Caliciviridae family the arrest was also conserved in rabbit hemorrhagic disease virus (RHDV) VPg and human sapovirus (HuSV) VPg. Truncation of MNV VPg shows that the first 62 amino acids are sufficient for a cell cycle arrest, and alignment of VPg sequences revealed a conserved motif in the N-terminal region of VPg. Analysis of VPg constructs with single N-terminal region point mutations, or exchange of N-terminal regions between VPg proteins, confirmed the importance of the N-terminal region for cell cycle arrest. These results provide evidence that G0/G1 cell cycle arrest is a conserved function of norovirus VPg proteins that involves the N-terminal region of these proteins.

Training: improving antenatal detection and outcomes of congenital heart disease.

OBJECTIVES: This study describes the design, delivery and efficacy of a regional fetal cardiac ultrasound training programme. This programme aimed to improve the antenatal detection of congenital heart disease (CHD) and its effect on fetal and postnatal outcomes. DESIGN SETTING AND PARTICIPANTS: This was a prospective study that compared antenatal CHD detection rates by professionals from 13 hospitals in Wales before and after engaging in our 'skills development programme'. Existing fetal cardiac practice and perinatal outcomes were continuously audited and progressive targets were set. The work was undertaken by the Welsh Fetal Cardiovascular Network, Antenatal Screening Wales (ASW), a superintendent sonographer and a fetal cardiologist. INTERVENTIONS: A core professional network was established, engaging all stakeholders (including patients, health boards, specialist commissioners, ASW, ultrasonographers, radiologists, obstetricians, midwives and paediatricians). A cardiac educational lead (midwife, superintendent sonographer, radiologist, obstetrician, or a fetal medicine specialist) was established in each hospital. A new cardiac anomaly screening protocol ('outflow tract view') was created and training on the new protocol was systematically delivered at each centre. Data were prospectively collected and outcomes were continuously audited: locally by the lead fetal cardiologist; regionally by the Congenital Anomaly Register and Information Service in Wales; and nationally by the National Institute for Cardiac Outcomes and Research (NICOR) in the UK. MAIN OUTCOME MEASURES: Patient satisfaction; improvements in individual sonographer skills, confidence and competency; true positive referral rate; local hospital detection rate; national detection rate of CHD; clinical outcomes of selected cardiac abnormalities; reduction of geographical health inequality; cost efficacy. RESULTS: High levels of patient satisfaction were demonstrated and the professional skill mix in each centre was improved. The confidence and competency of sonographers was enhanced. Each centre demonstrated a reduction in the false-positive referral rate and a significant increase in cardiac anomaly detection rate. According to the latest NICOR data, since implementing the new training programme Wales has sustained its status as UK lead for CHD detection. Health outcomes of children with CHD have improved, especially in cases of transposition of the great arteries (for which no perinatal mortality has been reported since 2008). Standardised care led to reduction of geographical health inequalities with substantial cost saving to the National Health Service due to reduced false-positive referral rates. Our successful model has been adopted by other fetal anomaly screening programmes in the UK. CONCLUSIONS: Antenatal cardiac ultrasound mass training programmes can be delivered effectively with minimal impact on finite healthcare resources. Sustainably high CHD detection rates can only be achieved by empowering the regional screening workforce through continuous investment in lifelong learning activities. These should be underpinned by high quality service standards, effective care pathways, and robust clinical governance and audit practices.

SMASH! The Salford medication safety dashboard.

BACKGROUND: Patient safety is vital to well-functioning health systems. A key component is safe prescribing, particularly in primary care where most medications are prescribed. Previous research demonstrated that the number of patients exposed to potentially hazardous prescribing can be reduced by interrogating the electronic health record (EHR) database of general practices and providing feedback to general practitioners in a pharmacist-led intervention. We aimed to develop and roll out an online dashboard application that delivers this audit and feedback intervention in a continuous fashion. METHOD: Based on initial system requirements we designed the dashboard's user interface over 3 iterations with 6 general practitioners (GPs), 7 pharmacists and a member of the public. Prescribing safety indicators from previous work were implemented in the dashboard. Pharmacists were trained to use the intervention and deliver it to general practices. RESULTS: A web-based electronic dashboard was developed and linked to shared care records in Salford, UK. The completed dashboard was deployed in all but one (n=43) general practices in the region. By November 2017, 36 pharmacists had been trained in delivering the intervention to practices. There were 135 registered users of the dashboard, with an average of 91 user sessions a week. CONCLUSION: We have developed and successfully rolled out of a complex, pharmacist-led dashboard intervention in Salford, UK. System usage statistics indicate broad and sustained uptake of the intervention. The use of systems that provide regularly updated audit information may be an important contributor towards medication safety in primary care.

Relationship between endotoxin core, staphylococcal and varicella antibody levels and outcome following aortic valve replacement surgery: a prospective observational study.

BACKGROUND: Morbidity and mortality following cardiac valve surgery is high. Immunity is an important contributor to outcome. This study examines the relationship of staphylococcal and endotoxin antibody levels to outcome following cardiac surgery. METHODS: Using enzyme-linked immunosorbent assays (ELISA), we measured pre-operative levels of antibodies to endotoxin core (EndoCAb); 3 common staphylococcal epitopes and varicella on saved serum of 60 adult patients scheduled to undergo elective primary surgical aortic valve replacement (AVR). Primary outcome measure was post-operative length of stay (LOS) in hospital with secondary outcomes being development of infective complications, length of stay on the intensive care unit (ICU) and 30-day mortality. Patients were quartiled according to antibody levels and outcomes compared between the quartile groups using Mann-Whitney tests for length of stay and Fisher's test for development of infection. RESULTS: Sixty patients (34 M, 26 F) were recruited with mean age 73 years (IQR 66-78), mean body mass index (BMI) 27.7 (IQR 25-31) and EuroSCORE II 1.44 (0.95-1.99). Those patients in the lower quartile for pre-operative antibody level had a longer post-operative stay than the upper quartile. EndoCAb (median IgG level Q1 42.2 MU/ml vs Q4 256 MU/ml) 9 vs 6 days, p = 0.025; alpha-toxin (median IgG level Q1 63 U vs Q4 558 U) 10 vs 7 days, p = 0.034; teichoic acid (median IgG level Q1 14 U vs Q4 419 U) 10 vs 8 days, p = 0.441; staphylococcal enterotoxin A (median IgG level Q1 55 U vs Q4 427 U) 9 vs 7 days, p = 0.865; varicella zoster (median IgG level Q1 1.325 U vs Q4 2.54 U) 8 vs 7 days, p = 1.0; and combined antibody levels 10 vs 6 days, p = 0.017. There were no differences in the number developing post-operative infections for each antibody type. The combined antibody analysis suggested a reduction in proportion of individuals developing infection from the upper vs lower quartile: 0 vs 0.33, p = 0.042. CONCLUSIONS: This study again suggests the inverse relationship between endotoxin core antibody levels and outcome following aortic valve surgery as well as suggesting a similar relationship with antibodies to staphylococcus. There is no such relationship for antibody levels against an organism not providing a peri-operative threat. Understanding this relationship may enable therapeutic manipulation of immune status, re-evaluation of risk and further investigation of the low immune state. TRIAL REGISTRATION: The patients in this study are a sub-group of the RELIEF AS study.ClinicalTrials.gov identifier NCT02174471.

High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms.

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

A Mass Spectrometry-Based Approach for Mapping Protein Subcellular Localization Reveals the Spatial Proteome of Mouse Primary Neurons.

We previously developed a mass spectrometry-based method, dynamic organellar maps, for the determination of protein subcellular localization and identification of translocation events in comparative experiments. The use of metabolic labeling for quantification (stable isotope labeling by amino acids in cell culture [SILAC]) renders the method best suited to cells grown in culture. Here, we have adapted the workflow to both label-free quantification (LFQ) and chemical labeling/multiplexing strategies (tandem mass tagging [TMT]). Both methods are highly effective for the generation of organellar maps and capture of protein translocations. Furthermore, application of label-free organellar mapping to acutely isolated mouse primary neurons provided subcellular localization and copy-number information for over 8,000 proteins, allowing a detailed analysis of organellar organization. Our study extends the scope of dynamic organellar maps to any cell type or tissue and also to high-throughput screening.

Expression of the NS5 (VPg) Protein of Murine Norovirus Induces a G1/S Phase Arrest.

Murine norovirus-1 (MNV-1) is known to subvert host cell division inducing an accumulation of cells in the G0/G1 phase, creating conditions where viral replication is favored. This study identified that NS5 (VPg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to MNV-1 infection. NS5 expression induced an accumulation of cells in the G0/G1 phase in an asynchronous population by inhibiting progression at the G1/S restriction point. Furthermore, NS5 expression resulted in a down-regulation of cyclin A expression in asynchronous cells and inhibited cyclin A expression in cells progressing from G1 to S phase. The activity of NS5 on the host cell cycle occurs through an uncharacterized function. Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G0/G1 phase as identified for wild-type NS5. To the best of our knowledge, this is the first report of a VPg protein manipulating the host cell cycle.

Variability of anti-staphylococcal antibodies in healthy volunteers and pre-cardiac surgery patients.

BACKGROUND: Pre-operative antibody levels have been shown to be inversely related to development of post-operative complications. Staphylococcal infection is a major source of morbidity following surgery. METHODS: We examined the variability of anti-staphylococcal antibody levels across a group of healthy volunteers and compared this with patients scheduled to undergo cardiac surgery. RESULTS: Pre-operative cardiac surgical patients exhibited significantly higher levels of staphylococcal antibodies compared with healthy volunteers. CONCLUSIONS: The relationship between pre-surgery staphylococcal antibody levels and outcome warrants further investigation.

Prevalence of defined ultrasound findings of unknown significance at the second trimester fetal anomaly scan and their association with adverse pregnancy outcomes: the Welsh study of mothers and babies population-based cohort.

OBJECTIVE: The aim of this article was to estimate the population prevalence of seven defined ultrasound findings of uncertain significance ('markers') in the second trimester and the associated risk of adverse pregnancy outcomes. METHOD: A prospective record-linked cohort study of 30 078 pregnant women who had second trimester anomaly scans between July 2008 and March 2011 in Wales was conducted. RESULTS: The prevalence of markers ranged from 43.7 per 1000 singleton pregnancies for cardiac echogenic foci [95% confidence interval (CI): 38.8, 51.1] to 0.6 for mild-to-moderate ventriculomegaly (95% CI: 0.3, 1.0). Isolated echogenic bowel was associated with an increased risk of congenital anomalies [risk ratio (RR) 4.54, 95% CI: 2.12, 9.73] and preterm birth (RR 2.30, 95% CI: 1.08, 4.90). Isolated pelvicalyceal dilatation was associated with an increased risk of congenital anomalies (RR 3.82, 95% CI: 2.16, 6.77). Multiple markers were associated with an increased risk of congenital anomalies (RR 5.00, 95% CI: 1.35, 18.40) and preterm birth (RR 3.38, 95% CI 1.20, 9.53). CONCLUSIONS: These data are useful for counselling families and developing clinical guidance and care pathways following the detection of markers in clinical practice, particularly the need for follow-up scans to monitor placental function and growth in pregnancies with isolated echogenic bowel, and further investigation for multiple markers. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.

Recanalisation and ulcer recurrence rates following ultrasound-guided foam sclerotherapy.

OBJECTIVES: The aim was to assess vein recanalisation and ulcer recurrence in patients with chronic venous ulceration following ultrasound-guided foam sclerotherapy. METHOD: Open (CEAP 6) or recently healed (CEAP 5) chronic venous leg ulcers were treated with foam sclerotherapy between July 2010 and August 2012. Venous duplex scans were performed two weeks and one and two years post treatment, recording recanalisation and reflux. One- and two-year ulcer recurrence rates were calculated using Kaplan-Meier survival analysis. RESULTS: A total of 100 legs were treated in 92 patients; 86 were CEAP 5 and 14 were CEAP 6. At two weeks complete or short segment occlusion was demonstrated in 99/100 legs. Complete follow-up was 93% at one year and 88% at two years. Complete and segmental recanalisation with new reflux was recorded in 11/93 (12%) legs and 25/93 (27%) legs, respectively (overall 36/93; 39%) at one year; and 6/88 (7%) legs and 15/88 (17%) legs, respectively (overall 21/88; 24%) at two years. Kaplan-Meier survival analysis shows 64% ulcers healed at 24 weeks and 86% at one year. Ulcer recurrence rate at one and two years was 2.3% and 5.1%. CONCLUSIONS: Over one-third of superficial veins treated with foam sclerotherapy recanalised at one year and just under a quarter of superficial veins recanalised at two years. Despite this, ulcer recurrence rates were low, and recanalisation failed to predict recurrence.

User Interface Requirements for Web-Based Integrated Care Pathways: Evidence from the Evaluation of an Online Care Pathway Investigation Tool.

Integrated care pathways (ICPs) define a chronological sequence of steps, most commonly diagnostic or treatment, to be followed in providing care for patients. Care pathways help to ensure quality standards are met and to reduce variation in practice. Although research on the computerisation of ICP progresses, there is still little knowledge on what are the requirements for designing user-friendly and usable electronic care pathways, or how users (normally health care professionals) interact with interfaces that support design, analysis and visualisation of ICPs. The purpose of the study reported in this paper was to address this gap by evaluating the usability of a novel web-based tool called COCPIT (Collaborative Online Care Pathway Investigation Tool). COCPIT supports the design, analysis and visualisation of ICPs at the population level. In order to address the aim of this study, an evaluation methodology was designed based on heuristic evaluations and a mixed method usability test. The results showed that modular visualisation and direct manipulation of information related to the design and analysis of ICPs is useful for engaging and stimulating users. However, designers should pay attention to issues related to the visibility of the system status and the match between the system and the real world, especially in relation to the display of statistical information about care pathways and the editing of clinical information within a care pathway. The paper concludes with recommendations for interface design.

Murine norovirus replication induces G0/G1 cell cycle arrest in asynchronously growing cells.

UNLABELLED: Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae. IMPORTANCE: Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication.

The Welsh study of mothers and babies: protocol for a population-based cohort study to investigate the clinical significance of defined ultrasound findings of uncertain significance.

BACKGROUND: Improvement in ultrasound imaging has led to the identification of subtle non-structural markers during the 18 - 20 week fetal anomaly scan, such as echogenic bowel, mild cerebral ventriculomegaly, renal pelvicalyceal dilatation, and nuchal thickening. These markers are estimated to occur in between 0.6% and 4.3% of pregnancies. Their clinical significance, for pregnancy outcomes or childhood morbidity, is largely unknown. The aim of this study is to estimate the prevalence of seven markers in the general obstetric population and establish a cohort of children for longer terms follow-up to assess the clinical significance of these markers. METHODS/DESIGN: All women receiving antenatal care within six of seven Welsh Health Boards who had an 18 to 20 week ultrasound scan in Welsh NHS Trusts between July 2008 and March 2011 were eligible for inclusion. Data were collected on seven markers (echogenic bowel, cerebral ventriculomegaly, renal pelvicalyceal dilatation, nuchal thickening, cardiac echogenic foci, choroid plexus cysts, and short femur) at the time of 18 - 20 week fetal anomaly scan. Ultrasound records were linked to routinely collected data on pregnancy outcomes (work completed during 2012 and 2013). Images were stored and reviewed by an expert panel.The prevalence of each marker (reported and validated) will be estimated. A projected sample size of 23,000 will allow the prevalence of each marker to be estimated with the following precision: a marker with 0.50% prevalence to within 0.10%; a marker with 1.00% prevalence to within 0.13%; and a marker with 4.50% prevalence to within 0.27%. The relative risk of major congenital abnormalities, stillbirths, pre-term birth and small for gestational age, given the presence of a validated marker, will be reported. DISCUSSION: This is a large, prospective study designed to estimate the prevalence of markers in a population-based cohort of pregnant women and to investigate associations with adverse pregnancy outcomes. The study will also establish a cohort of children that can be followed-up to explore associations between specific markers and longer-term health and social outcomes.

Verbal protocols for assessing the usability of clinical decision support: the retrospective sense making protocol.

We compare the effectiveness of two types of verbal protocol, concurrent think aloud vs. retrospective sense making, for evaluating the usability of a clinical decision support tool. Thirty-five medical and nursing students participated in a usability experiment. Participants were asked to complete seven tasks using the system under evaluation. Eighteen students were allocated to the concurrent think aloud group and the remainder followed the retrospective protocol. The retrospective protocol was significantly more sensitive than the concurrent protocol in recording unique usability problems related to users' cognitive behaviour. These problems concerned the interpretation and comprehension of statistical output, search results and system messages. These findings can be explained by the retrospective protocol's greater ability to detect compound usability problems, capturing the cognitive dimensions of users' interactions with the interface in greater depth. Evaluations of clinical decision support systems should take an evidence-based approach to selecting protocols.

Variations in the anatomical distribution of peripheral vascular disease according to gender.

BACKGROUND: While large epidemiological studies have suggested that the male gender is more frequently afflicted with intermittent claudication, there is little data whether there are gender differences in the distribution of peripheral vascular disease (PVD). The aim of this study was to clarify this issue on the basis of angiographic findings in patients presenting with claudication. PATIENTS AND METHODS: The radiology department computerised database was used to identify all lower limb angiograms performed for investigation of PVD. Patients undergoing incomplete assessment and those with normal angiograms were excluded. Demographic details for each patient were collected together with details of uni- or bilaterality of disease, the number of lesions present and their anatomical distribution according to the major named vessels. Only lesions reported as radiologically significant were included in the analysis. RESULTS: Five hundred consecutive angiograms fitting the defined study criteria were assessed. There were 310 males and 190 females giving a male to female ratio of 1.6:1. The most common distribution for both genders was multiple bilateral lesions. There were no significant differences in the number of stenoses in terms of ratio of bilateral to unilateral (2.39 vs 2.77) or ratio of multiple to single lesions (1.5 vs 1.7) between the female and male groups. Disease was more common in males at all anatomical locations, the most significant differences being for lesions of the common femoral and profunda femoris arteries. CONCLUSIONS: PVD is more commonly diagnosed in males than females. The disease process is more commonly bilateral in both genders and PVD affects more numerous sites in the male claudicant than in the female claudicant. Nevertheless, there does not appear to be any difference in the anatomical distribution of disease between genders.

Trace-Level Automated Mercury Speciation Analysis.

An automated system for methyl Hg analysis by purge and trap gas chromatography (GC) was evaluated, with comparison of several different instrument configurations including chromatography columns (packed column or capillary), detector (atomic fluorescence, AFS, or inductively coupled plasma mass spectrometry, ICP-MS, using quadrupole and sector field ICP- MS instruments). Method detection limits (MDL) of 0.042 pg and 0.030 pg for CH(3)Hg(+) were achieved with the automated Hg analysis system configured with AFS and ICPMS detection, respectively. Capillary GC with temperature programming was effective in improving resolution and decreasing retention times of heavier Hg species (in this case C(3)H(7)Hg(+)) although carryover between samples was increased. With capillary GC, the MDL for CH(3)Hg(+) was 0.25 pg for AFS detection and 0.060 pg for ICP-MS detection. The automated system was demonstrated to have high throughput (72 samples analyzed in 8 hours) requiring considerably less analyst time than the manual method for methyl mercury analysis described in EPA 1630.