Comparison of in-gel and in-solution proteolysis in the proteome profiling of organ perfusion solutions.

PURPOSE: The organ perfusion solution (perfusate), collected at clinically and temporally significant stages of the organ preservation and transplantation process, provides a valuable insight into the biological status of an organ over time and prior to reperfusion (transplantation) in the recipient. The objective of this study was to assess two bottom-up proteomics workflows for the extraction of tryptic peptides from the perfusate. EXPERIMENTAL DESIGN: Two different kinds of perfusate samples from kidney and liver trials were profiled using liquid chromatography-mass spectrometry (LC-MS/MS). The preparation of clean peptide mixtures for downstream analysis was performed considering different aspects of sample preparation; protein estimation, enrichment, in-gel and urea-based in-solution digestion. RESULTS: In-solution digestion of perfusate allowed identification of the highest number of peptides and proteins with greater sequence coverage and higher confidence data in kidney and liver perfusate. Key pathways identified by gene ontology analysis included complement, coagulation and antioxidant pathways, and a number of biomarkers previously linked to ischemia-reperfusion injury were also observed in perfusate. CONCLUSIONS: This study showed that in-solution digestion is a more efficient method for LC-MS/MS analysis of kidney and liver organ perfusion solutions. This method is also quicker and easier than in-gel digestion, allowing for greater sample throughput, with fewer opportunities for experimental error or peptide loss.

Determining the sample size for a cluster-randomised trial using knowledge elicitation: Bayesian hierarchical modelling of the intracluster correlation coefficient.

BACKGROUND: The intracluster correlation coefficient is a key input parameter for sample size determination in cluster-randomised trials. Sample size is very sensitive to small differences in the intracluster correlation coefficient, so it is vital to have a robust intracluster correlation coefficient estimate. This is often problematic because either a relevant intracluster correlation coefficient estimate is not available or the available estimate is imprecise due to being based on small-scale studies with low numbers of clusters. Misspecification may lead to an underpowered or inefficiently large and potentially unethical trial. METHODS: We apply a Bayesian approach to produce an intracluster correlation coefficient estimate and hence propose sample size for a planned cluster-randomised trial of the effectiveness of a systematic voiding programme for post-stroke incontinence. A Bayesian hierarchical model is used to combine intracluster correlation coefficient estimates from other relevant trials making use of the wealth of intracluster correlation coefficient information available in published research. We employ knowledge elicitation process to assess the relevance of each intracluster correlation coefficient estimate to the planned trial setting. The team of expert reviewers assigned relevance weights to each study, and each outcome within the study, hence informing parameters of Bayesian modelling. To measure the performance of experts, agreement and reliability methods were applied. RESULTS: The 34 intracluster correlation coefficient estimates extracted from 16 previously published trials were combined in the Bayesian hierarchical model using aggregated relevance weights elicited from the experts. The intracluster correlation coefficients available from external sources were used to construct a posterior distribution of the targeted intracluster correlation coefficient which was summarised as a posterior median with a 95% credible interval informing researchers about the range of plausible sample size values. The estimated intracluster correlation coefficient determined a sample size of between 450 (25 clusters) and 480 (20 clusters), compared to 500-600 from a classical approach. The use of quantiles, and other parameters, from the estimated posterior distribution is illustrated and the impact on sample size described. CONCLUSION: Accounting for uncertainty in an unknown intracluster correlation coefficient, trials can be designed with a more robust sample size. The approach presented provides the possibility of incorporating intracluster correlation coefficients from various cluster-randomised trial settings which can differ from the planned study, with the difference being accounted for in the modelling. By using expert knowledge to elicit relevance weights and synthesising the externally available intracluster correlation coefficient estimates, information is used more efficiently than in a classical approach, where the intracluster correlation coefficient estimates tend to be less robust and overly conservative. The intracluster correlation coefficient estimate constructed is likely to produce a smaller sample size on average than the conventional strategy of choosing a conservative intracluster correlation coefficient estimate. This may therefore result in substantial time and resources savings.

Clinical and cost effectiveness of arthritis gloves in rheumatoid arthritis (A-GLOVES): randomised controlled trial with economic analysis.

BACKGROUND: Arthritis (or compression) gloves are widely prescribed to people with rheumatoid arthritis and other forms of hand arthritis. They are prescribed for daytime wear to reduce hand pain and improve hand function, and/or night-time wear to reduce pain, improve sleep and reduce morning stiffness. However, evidence for their effectiveness is limited. The aims of this study were to investigate the clinical and cost effectiveness of arthritis gloves compared to placebo gloves on hand pain, stiffness and function in people with rheumatoid arthritis and persistent hand pain. METHODS: A parallel randomised controlled trial, in adults (≥ 18 years) with rheumatoid or undifferentiated inflammatory arthritis at 16 National Health Service sites in the UK. Patients with persistent hand pain affecting function and/or sleep were eligible. Randomisation (1:1) was stratified by recent change (or not) in medication, using permuted blocks of random sizes. Three-quarter-finger length arthritis gloves (Isotoner®: applying 23-32 mmHg pressure) (intervention) were compared to loose-fitting placebo gloves (Jobskin® classic: providing no/minimal pressure) (control). Both gloves (considered to have similar thermal qualities) were provided by occupational therapists. Patients and outcome assessors were blinded; clinicians were not. The primary outcome was dominant hand pain on activity (0-10) at 12 weeks, analysed using linear regression and intention to treat principles. RESULTS: Two hundred six participants were randomly assigned (103 per arm) and 163 (84 intervention: 79 control) completed 12-week follow-up. Hand pain improved by 1.0 (intervention) and 1.2 (control), an adjusted mean difference of 0.10 (95% CI: - 0.47 to 0.67; p = 0.72). Adverse events were reported by 51% of intervention and 36% of control group participants; with 6 and 7% respectively, discontinuing glove wear. Provision of arthritis gloves cost £129, with no additional benefit. CONCLUSION: The trial provides evidence of no clinically important effect of arthritis gloves on any of the trial outcomes (hand pain, function and stiffness) and arthritis gloves are not cost-effective. The clinical and cost-effectiveness results support ceasing provision of arthritis gloves in routine clinical practice. FUNDING: National Institute for Health Research. TRIAL REGISTRATION: ISRCTN, ISRCTN25892131 ; Registered 05/09/2016: retrospectively registered.

Journey of vulnerability: a mixed-methods study to understand intrapartum transfers in Tanzania and Zambia.

BACKGROUND: Timely intrapartum referral between facilities is pivotal in reducing maternal/neonatal mortality and morbidity but is distressing to women, resource-intensive and likely to cause delays in care provision. We explored the complexities around referrals to gain understanding of the characteristics, experiences and outcomes of those being transferred. METHODS: We used a mixed-method parallel convergent design, in Tanzania and Zambia. Quantitative data were collected from a consecutive, retrospective case-note review (target, n = 2000); intrapartum transfers and stillbirths were the outcomes of interest. A grounded theory approach was adopted for the qualitative element; data were collected from semi-structured interviews (n = 85) with women, partners and health providers. Observations (n = 33) of transfer were also conducted. Quantitative data were analysed descriptively, followed by binary logistic regression models, with multiple imputation for missing data. Qualitative data were analysed using Strauss's constant comparative approach. RESULTS: Intrapartum transfer rates were 11% (111/998; 2 unknown) in Tanzania and 37% (373/996; 1 unknown) in Zambia. Main reasons for transfer were prolonged/obstructed labour and pre-eclampsia/eclampsia. Women most likely to be transferred were from Zambia (as opposed to Tanzania), HIV positive, attended antenatal clinic < 4 times and living > 30 min away from the referral hospital. Differences were observed between countries. Of those transferred, delays in care were common and an increase in poor outcomes was observed. Qualitative findings identified three categories: social threats to successful transfer, barriers to timely intrapartum care and reparative interventions which were linked to a core category: journey of vulnerability. CONCLUSION: Although intrapartum transfers are inevitable, modifiable factors exist with the potential to improve the experience and outcomes for women. Effective transfers rely on adequate resources, effective transport infrastructures, social support and appropriate decision-making. However, women's (and families) vulnerability can be reduced by empathic communication, timely assessment and a positive birth outcome; this can improve women's resilience and influence positive decision-making, for the index and future pregnancy.

IMPlementation of An online Relatives' Toolkit for psychosis or bipolar (IMPART study): iterative multiple case study to identify key factors impacting on staff uptake and use.

BACKGROUND: Despite the potential of digital health interventions to improve the delivery of psychoeducation to people with mental health problems and their relatives, and substantial investment in their development, there is little evidence of successful implementation into clinical practice. We report the first implementation study of a digital health intervention: Relatives Education And Coping Toolkit (REACT), into routine mental healthcare. Our main aim was to identify critical factors affecting staff uptake and use of this online self-management tool for relatives of people with psychosis or bipolar. METHODS: A mixed-methods, theory-driven (Normalisation Process Theory), iterative multiple case study approach using qualitative analysis of interviews with staff and quantitative reporting of uptake. Carer researchers were part of the research team. RESULTS: In all, 281 staff and 159 relatives from Early Intervention teams across six catchment areas (cases) in England registered on REACT; 129 staff took part in qualitative interviews. Staff were positive about REACT helping services improve support and meet clinical targets. Implementation was hindered by: high staff caseloads and difficulties prioritising carers; perception of REACT implementation as research; technical difficulties using REACT; poor interoperability with trust computer systems and care pathways; lack of access to mobile technology and training; restricted forum populations; staff fears of risk, online trolling, and replacement by technology; and uncertainty around REACT's long-term availability. CONCLUSIONS: Digital health interventions, such as REACT, should be iteratively developed, evaluated, adapted and implemented, in partnership with the services they aim to support, and as part of a long term national strategy to co-develop integrated technology-enabled mental healthcare. Implementation strategies must instil a sense of ownership for staff and ensure they have adequate IT training, appropriate governance protocols for online working, and adequate mobile technologies. Wider contextual factors including adequate funding for mental health services and prioritisation of carer support, also need to be addressed for successful implementation of carer focussed digital interventions. TRIAL REGISTRATION: Study registration: ISCTRN 16267685.

The effect on work presenteeism of job retention vocational rehabilitation compared to a written self-help work advice pack for employed people with inflammatory arthritis: protocol for a multi-centre randomised controlled trial (the WORKWELL trial).

BACKGROUND: Work problems are common in people with inflammatory arthritis. Up to 50% stop work within 10 years due to their condition and up to 67% report presenteeism (i.e. reduced work productivity), even amongst those with low disease activity. Job retention vocational rehabilitation (JRVR) may help prevent or postpone job loss and reduce presenteeism through work assessment, work-related rehabilitation and enabling job accommodations. This aims to create a better match between the person's abilities and their job demands. The objectives of the Workwell trial are to test the overall effectiveness and cost-effectiveness of JRVR (WORKWELL) provided by additionally trained National Health Service (NHS) occupational therapists compared to a control group who receive self-help information both in addition to usual care. METHODS: Based on the learning from a feasibility trial (the WORK-IA trial: ISRCTN76777720 ), the WORKWELL trial is a multi-centre, pragmatic, individually-randomised parallel group superiority trial, including economic evaluation, contextual factors analysis and process evaluation. Two hundred forty employed adults with rheumatoid arthritis, undifferentiated inflammatory arthritis or psoriatic arthritis (in secondary care), aged 18 years or older with work instability will be randomised to one of two groups: a self-help written work advice pack plus usual care (control intervention); or WORKWELL JRVR plus a self-help written work advice pack and usual care. WORKWELL will be delivered by occupational therapists provided with additional JRVR training from the research team. The primary outcome is presenteeism as measured using the Work Limitations Questionnaire-25. A comprehensive range of secondary outcomes of work, health, contextual factors and health resource use are included. Outcomes are measured at 6- and 12- months (with 12-months as the primary end-point). A multi-perspective within-trial cost-effectiveness analyses will also be conducted. DISCUSSION: This trial will contribute to the evidence base for provision of JRVR to people with inflammatory arthritis. If JRVR is found to be effective in enabling people to keep working, the findings will support decision-making about provision of JRVR by rheumatology teams, therapy services and healthcare commissioners, and providing evidence of the effectiveness of JRVR and the economic impact of its implementation. TRIAL REGISTRATION: Clinical Trials.Gov: NCT03942783 . Registered 08/05/2019 ( https://clinicaltrials.gov/ct2/show/NCT03942783 ); ISRCTN Registry: ISRCTN61762297 . Registered:13/05/2019 ( http://www.isrctn.com/ISRCTN61762297 ). Retrospectively registered.

The effectiveness and cost-effectiveness of strength and balance Exergames to reduce falls risk for people aged 55 years and older in UK assisted living facilities: a multi-centre, cluster randomised controlled trial.

BACKGROUND: Falls are the leading cause of fatal and non-fatal unintentional injuries in older people. The use of Exergames (active, gamified video-based exercises) is a possible innovative, community-based approach. This study aimed to determine the effectiveness of a tailored OTAGO/FaME-based strength and balance Exergame programme for improving balance, maintaining function and reducing falls risk in older people. METHODS: A two-arm cluster randomised controlled trial recruiting adults aged 55 years and older living in 18 assisted living (sheltered housing) facilities (clusters) in the UK. Standard care (physiotherapy advice and leaflet) was compared to a tailored 12-week strength and balance Exergame programme, supported by physiotherapists or trained assistants. Complete case analysis (intention-to-treat) was used to compare the Berg Balance Scale (BBS) at baseline and at 12 weeks. Secondary outcomes included fear of falling, mobility, fall risk, pain, mood, fatigue, cognition, healthcare utilisation and health-related quality of life, and self-reported physical activity and falls. RESULTS: Eighteen clusters were randomised (9 to each arm) with 56 participants allocated to the intervention and 50 to the control (78% female, mean age 78 years). Fourteen participants withdrew over the 12 weeks (both arms), mainly for ill health. There was an adjusted mean improvement in balance (BBS) of 6.2 (95% CI 2.4 to 10.0) and reduced fear of falling (p = 0.007) and pain (p = 0.02) in the Exergame group. Mean attendance at sessions was 69% (mean exercising time of 33 min/week). Twenty-four percent of the control group and 20% of the Exergame group fell over the trial period. The change in fall rates significantly favoured the intervention (incident rate ratio 0.31 (95% CI 0.16 to 0.62, p = 0.001)). The point estimate of the incremental cost-effectiveness ratio (ICER) was £15,209.80 per quality-adjusted life year (QALY). Using 10,000 bootstrap replications, at the lower bound of the NICE threshold of £20,000 per QALY, there was a 61% probability of Exergames being cost-effective, rising to 73% at the upper bound of £30,000 per QALY. CONCLUSIONS: Exergames, as delivered in this trial, improve balance, pain and fear of falling and are a cost-effective fall prevention strategy in assisted living facilities for people aged 55 years or older. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov on 18 Dec 2015 with reference number NCT02634736 .

Proteomics analysis of colon cancer progression.

BACKGROUND: The aim of this pilot study was to identify proteins associated with advancement of colon cancer (CC). METHODS: A quantitative proteomics approach was used to determine the global changes in the proteome of primary colon cancer from patients with non-cancer normal colon (NC), non-adenomatous colon polyp (NAP), non-metastatic tumor (CC NM) and metastatic tumor (CC M) tissues, to identify up- and down-regulated proteins. Total protein was extracted from each biopsy, trypsin-digested, iTRAQ-labeled and the resulting peptides separated using strong cation exchange (SCX) and reverse-phase (RP) chromatography on-line to electrospray ionization mass spectrometry (ESI-MS). RESULTS: Database searching of the MS/MS data resulted in the identification of 2777 proteins which were clustered into groups associated with disease progression. Proteins which were changed in all disease stages including benign, and hence indicative of the earliest molecular perturbations, were strongly associated with spliceosomal activity, cell cycle division, and stromal and cytoskeleton disruption reflecting increased proliferation and expansion into the surrounding healthy tissue. Those proteins changed in cancer stages but not in benign, were linked to inflammation/immune response, loss of cell adhesion, mitochondrial function and autophagy, demonstrating early evidence of cells within the nutrient-poor solid mass either undergoing cell death or adjusting for survival. Caveolin-1, which decreased and Matrix metalloproteinase-9, which increased through the three disease stages compared to normal tissue, was selected to validate the proteomics results, but significant patient-to-patient variation obfuscated interpretation so corroborated the contradictory observations made by others. CONCLUSION: Nevertheless, the study has provided significant insights into CC stage progression for further investigation.

Knee problems are common in young adults and associated with physical activity and not obesity: the findings of a cross-sectional survey in a university cohort.

BACKGROUND: Obesity and sedentary behaviour, risk factors for knee osteoarthritis in middle-age, are increasing in younger adults. The objectives of this study were to estimate the prevalence of knee problems in young adults, to characterise these problems and explore the relationship with physical activity, physical inactivity and obesity. METHODS: Presence of knee problems was collected through self-report questionnaire from staff and students of one university aged 18-39; direct measurement of weight and height was taken and activity measured using the International Physical Activity Questionnaire. Twelve-month prevalence of knee problems was estimated. Logistic regression was used to investigate the relationship between knee problems and physical activity levels, sitting time and body mass index. RESULTS: The prevalence of knee problems was high (31.8% [95% CI 26.9 to 37.2%]) among the 314 participants; knee pain was the most common dominant symptom (65%). Only high physical activity levels (OR 2.6 [95% CI 1.4-4.9]) and mental distress (OR 2.3 [95% CI 1.2-4.6]) were independent risk factors for knee problems. CONCLUSIONS: Knee problems were common among young adults, who were staff and students of a university. With increasing obesity prevalence, populations are being encouraged to become more active. More attention may need to be paid towards prevention of knee problems in such programmes, and further research is warranted.

Evaluation of nipple aspirate fluid as a diagnostic tool for early detection of breast cancer.

There has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancer-related deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of "omics" strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed.

The effects of arthritis gloves on people with Rheumatoid Arthritis or Inflammatory Arthritis with hand pain: a study protocol for a multi-centre randomised controlled trial (the A-GLOVES trial).

BACKGROUND: Arthritis gloves are regularly provided as part of the management of people with rheumatoid arthritis (RA) and undifferentiated (early) inflammatory arthritis (IA). Usually made of nylon and elastane (i.e. Lycra®), these arthritis gloves apply pressure with the aims of relieving hand pain, stiffness and improving hand function. However, a systematic review identified little evidence supporting their use. We therefore designed a trial to compare the effectiveness of the commonest type of arthritis glove provided in the United Kingdom (Isotoner gloves) (intervention) with placebo (control) gloves (i.e. larger arthritis gloves providing similar warmth to the intervention gloves but minimal pressure only) in people with these conditions. METHODS: Participants aged 18 years and over with RA or IA and persistent hand pain will be recruited from National Health Service Trusts in the United Kingdom. Following consent, participants will complete a questionnaire booklet, then be randomly allocated to receive intervention or placebo arthritis gloves. Within three weeks, they will be fitted with the allocated gloves by clinical specialist rheumatology occupational therapists. Twelve weeks (i.e. the primary endpoint) after completing the baseline questionnaire, participants will complete a second questionnaire, including the same measures plus additional questions to explore adherence, benefits and problems with glove-wear. A sub-sample of participants from each group will be interviewed at the end of their participation to explore their views of the gloves received. The clinical effectiveness and cost-effectiveness of the intervention, compared to placebo gloves, will be evaluated over 12 weeks. The primary outcome measure is hand pain during activity. Qualitative interviews will be thematically analysed. DISCUSSION: This study will evaluate the commonest type of arthritis glove (Isotoner) provided in the NHS (i.e. the intervention) compared to a placebo glove. The results will help occupational therapists, occupational therapy services and people with arthritis make informed choices as to the value of arthritis gloves. If effective, arthritis gloves should become more widely available in the NHS to help people with RA and IA manage hand symptoms and improve performance of daily activities, work and leisure. If not, services can determine whether to cease supplying these to reduce service costs. TRIAL REGISTRATION: ISRCTN Registry: ISRCTN25892131 Registered 05/09/2016.

A questionnaire to identify patellofemoral pain in the community: an exploration of measurement properties.

BACKGROUND: Community-based studies of patellofemoral pain (PFP) need a questionnaire tool that discriminates between those with and those without the condition. To overcome these issues, we have designed a self-report questionnaire which aims to identify people with PFP in the community. STUDY DESIGNS: comparative study and cross-sectional study. STUDY POPULATION: comparative study: PFP patients, soft-tissue injury patients and adults without knee problems. Cross-sectional study: adults attending a science festival. INTERVENTION: comparative study participants completed the questionnaire at baseline and two weeks later. Cross-sectional study participants completed the questionnaire once. The optimal scoring system and threshold was explored using receiver operating characteristic curves, test-retest reliability using Cohen's kappa and measurement error using Bland-Altman plots and standard error of measurement. Known-group validity was explored by comparing PFP prevalence between genders and age groups. RESULTS: Eighty-four participants were recruited to the comparative study. The receiver operating characteristic curves suggested limiting the questionnaire to the clinical features and knee pain map sections (AUC 0.97 95 % CI 0.94 to 1.00). This combination had high sensitivity and specificity (over 90 %). Measurement error was less than the mean difference between the groups. Test-retest reliability estimates suggest good agreement (N = 51, k = 0.74, 95 % CI 0.52-0.91). The cross-sectional study (N = 110) showed expected differences between genders and age groups but these were not statistically significant. CONCLUSION: A shortened version of the questionnaire, based on clinical features and a knee pain map, has good measurement properties. Further work is needed to validate the questionnaire in community samples.

Are there three main subgroups within the patellofemoral pain population? A detailed characterisation study of 127 patients to help develop targeted intervention (TIPPs).

BACKGROUND: Current multimodal approaches for the management of non-specific patellofemoral pain are not optimal, however, targeted intervention for subgroups could improve patient outcomes. This study explores whether subgrouping of non-specific patellofemoral pain patients, using a series of low cost simple clinical tests, is possible. METHOD: The exclusivity and clinical importance of potential subgroups was assessed by applying à priori test thresholds (1 SD) from seven clinical tests in a sample of adult patients with non-specific patellofemoral pain. Hierarchical clustering and latent profile analysis, were used to gain additional insights into subgroups using data from the same clinical tests. RESULTS: 130 participants were recruited, 127 had complete data: 84 (66%) female, mean age 26 years (SD 5.7) and mean body mass index 25.4 (SD 5.83), median (IQR) time between onset of pain and assessment was 24 (7-60) months. Potential subgroups defined by the à priori test thresholds were not mutually exclusive and patients frequently fell into multiple subgroups. Using hierarchical clustering and latent profile analysis three subgroups were identified using 6 of the 7 clinical tests. These subgroups were given the following nomenclature: (1) 'strong', (2) 'weak and tighter' and (3) 'weak and pronated foot'. CONCLUSIONS: We conclude that three subgroups of patellofemoral patients may exist based on the results of six clinical tests which are feasible to perform in routine clinical practice. Further research is needed to validate these findings in other data sets and, if supported by external validation, to see if targeted interventions for these subgroups improve patient outcomes.

Thin-layer chromatography/matrix-assisted laser desorption/ionisation mass spectrometry and matrix-assisted laser desorption/ionisation mass spectrometry imaging for the analysis of phospholipids in LS174T colorectal adenocarcinoma xenografts treated with the vascular disrupting agent DMXAA.

RATIONALE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a low molecular weight drug of the flavonoid group, which has an anti-vascular effect in tumours causing endothelial cell apoptosis and activation of cytokines. Flavonoid-based compounds have been reported to lead to an upregulation in the expression of lysophosphatidylcholines (LPC)-type lipids in solid tumours. A study employing TLC/MALDI-MS and MALDI-MS imaging to examine LS174T colorectal adenocarcinoma xenografts following administration of DMXAA has been conducted into this effect. METHODS: LS174T colorectal adenocarcinoma xenografts grown in male immune-deficient mice were treated with 27.5 mg/kg DMXAA. The control (before treatment) and 4 h and 24 h post-treatment tumours were excised and divided into two. MALDI-MS imaging experiments were carried out on 12 µm cryosections sections taken from one half of the tumours and from the other half the lipids were extracted and analysed by TLC/MALDI-MS. These experiments were carried out in triplicate. RESULTS: Statistical analysis of the MALDI-MS imaging data set indicated an increased amount of LPC in the 24 h post-treated sample and a decreased amount of PC in the 24 h post-treated sample, compared with the 4 h post-treated sample and the control. These effects were confirmed by the TLC/MALDI-MS data. The lipid extracts were separated into six spots on the TLC plate. These were identified as arising from different lipids classes, i.e. LPC, sphingomyelins (SM), phosphatidylcholines (PC) and phosphatidylethanolamines (PE). The TLC/MALDI-MS data indicated that LPC were highly expressed in the 4 h and 24 h post-treated tumour samples compared with the control. Examination of the mass spectrometric images confirms this increase and demonstrates additionally that the increase in the signals arising from LPC appears to be localised primarily within the central areas of the xenograft. CONCLUSIONS: An increase in expression of LPC lipids in solid tumours treated with DMXAA has been demonstrated and shown to be localised in the central area of the tumour.

Phenotypic consequences in black South African Fanconi anemia patients homozygous for a founder mutation.

PURPOSE: Fanconi anemia is a genotypically and phenotypically heterogeneous condition, characterized microscopically by chromosomal instability and breakage. Affected individuals manifest growth restriction and congenital physical abnormalities; most progress to hematological disease including bone marrow aplasia. Black South African Fanconi anemia patients share a common causative founder mutation in the Fanconi G gene in 80% of cases (637_643delTACCGCC). The aim of this study was to investigate the genotype-physical phenotype correlation in a cohort of individuals homozygous for this mutation. METHODS: Thirty-five black patients were recruited from tertiary level hematology/oncology clinics in South Africa. Participants were subjected to a comprehensive clinical examination, documenting growth, congenital anomalies, and phenotypic variability. RESULTS: Descriptive statistical analysis showed significant growth abnormalities in many patients and a high frequency (97%) of skin pigmentary anomalies. Subtle anomalies of the eyes, ears, and hands occurred frequently (≥70%). Apart from malformations of the kidney (in 37%) and gastrointestinal tract (in 8.5%), congenital anomalies of other systems including the cardiovascular and central nervous systems, genitalia, and vertebrae were infrequent (<5%). CONCLUSION: The diagnosis of Fanconi anemia in black South African patients before the onset of hematological symptoms remains a clinical challenge, with the physical phenotype unlikely to be recognized by those without dysmorphology expertise.

Patterns of cancer cell sphere formation in primary cultures of human oral tongue squamous cell carcinoma and neck nodes.

Recently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells.

Comprehensive understanding of the role of GPER in estrogen receptor-alpha negative breast cancer.

G protein-coupled estrogen receptor (GPER) plays a prominent role in facilitating the rapid, non-genomic signaling of estrogens in breast cancer cells. Herein, a comprehensive overview of the role of GPER in ER-ɑ-negative breast cancer is provided. Activation of GPER affected proliferation, metastasis and epithelial mesenchymal transition in ER-ɑ negative breast cancer cells. Clinical studies have demonstrated that GPER positivity was strongly correlated with larger tumor size and advanced clinical stage, suggesting that GPER/ERK signaling may play a role in promoting tumor progression. Strong evidence existed that environmental contaminants like bisphenol A have a carcinogenic potential mediated by GPER activation. The complexity of the cross talk between GPER and other receptors including ER-ß, ER-α36, Estrogen-related receptor α (ERRα) and androgen receptor has been discussed. The potential utility of small molecules and phytoestrogens targeting GPER, adds valuable insights into its therapeutic potential. This review holds promises in advancing our understanding of GPER role in ER-ɑ-negative breast cancer. Overall, the consequences of GPER activation are still an area of active research and the implication are not entirely clear.

Identification of stage-specific breast markers using quantitative proteomics.

Matched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.

Training emergency services' dispatchers to recognise stroke: an interrupted time-series analysis.

BACKGROUND: Stroke is a time-dependent medical emergency in which early presentation to specialist care reduces death and dependency. Up to 70% of all stroke patients obtain first medical contact from the Emergency Medical Services (EMS). Identifying 'true stroke' from an EMS call is challenging, with over 50% of strokes being misclassified. The aim of this study was to evaluate the impact of the training package on the recognition of stroke by Emergency Medical Dispatchers (EMDs). METHODS: This study took place in an ambulance service and a hospital in England using an interrupted time-series design. Suspected stroke patients were identified in one week blocks, every three weeks over an 18 month period, during which time the training was implemented. Patients were included if they had a diagnosis of stroke (EMS or hospital). The effect of the intervention on the accuracy of dispatch diagnosis was investigated using binomial (grouped) logistic regression. RESULTS: In the Pre-implementation period EMDs correctly identified 63% of stroke patients; this increased to 80% Post-implementation. This change was significant (p=0.003), reflecting an improvement in identifying stroke patients relative to the Pre-implementation period both the During-implementation (OR=4.10 [95% CI 1.58 to 10.66]) and Post-implementation (OR=2.30 [95% CI 1.07 to 4.92]) periods. For patients with a final diagnosis of stroke who had been dispatched as stroke there was a marginally non-significant 2.8 minutes (95% CI -0.2 to 5.9 minutes, p=0.068) reduction between Pre- and Post-implementation periods from call to arrival of the ambulance at scene. CONCLUSIONS: This is the first study to develop, implement and evaluate the impact of a training package for EMDs with the aim of improving the recognition of stroke. Training led to a significant increase in the proportion of stroke patients dispatched as such by EMDs; a small reduction in time from call to arrival at scene by the ambulance also appeared likely. The training package has been endorsed by the UK Stroke Forum Education and Training, and is free to access on-line.