The Finnish Diabetes Prevention Study (DPS) was a randomized controlled trial, which showed that it is possible to prevent type 2 diabetes by lifestyle changes. The aim of the present study was to examine whether the lifestyle intervention had an effect on the ten-year mortality and cardiovascular morbidity in the DPS participants originally randomized either into an intervention or control group. Furthermore, we compared these results with a population-based cohort comprising individuals of varying glucose tolerance states.Middle-aged, overweight people with IGT (n = 522) were randomized into intensive intervention , or control “mini-intervention” group. Median length of the intervention period was 4 years and the mean follow-up was 10.6 years. The population-based reference study cohort included 1881 individuals  with the mean follow-up of 13.8 years. Mortality and cardiovascular morbidity data were collected from the national Hospital Discharge Register and Causes of Death Register. Among the DPS participants who consented for register linkage (n = 505), total mortality  and cardiovascular morbidity  did not differ significantly between the intervention and control groups. Compared with the population-based cohort with impaired glucose tolerance, adjusted HRs were 0.21 (95% CI 0.09–0.52) and 0.39 (95% CI 0.20–0.79) for total mortality, and 0.89 (95% CI 0.62–1.27) and 0.87 (0.60–1.27) for cardiovascular morbidity in the intervention and control groups of the DPS, respectively. The risk of death in DPS combined cohort was markedly lower than in FINRISK IGT cohort , but there was no significant difference in the risk of CVD .Lifestyle intervention among persons with IGT did not decrease cardiovascular morbidity during the first 10 years of follow-up. However, the statistical power may not be sufficient to detect small differences between the intervention and control groups. Low total mortality among participants of the DPS compared with individuals with IGT in the general population could be ascribed to a lower cardiovascular risk profile at baseline and regular follow-up.NCT00518167ClinicalTrials.gov  Type 2 diabetes mellitus (T2DM) as well as lesser degrees of impaired glucose regulation are known to increase the risk of cardiovascular diseases (CVD), in particular coronary heart disease, peripheral vascular diseases, and stroke However, the ultimate goal of prevention of T2DM is to improve the prognosis and overall quality of life of affected individuals, and with regard to health policy, also to prevent increasing costs of the treatment of T2DM and its complications. There are no randomized lifestyle intervention trials in persons with IGT to show that lifestyle changes would reduce CVD mortality or morbidity. Recently, the 20-year results from the China Da Qing Diabetes Prevention Study The main aim of the present study was to examine whether the lifestyle intervention applied in the DPS had an effect on mortality and CVD morbidity by comparing the mortality and CVD morbidity data of the intensive lifestyle intervention group to control group. In addition, we also present follow-up data for a population-based “native control group” (the FINRISK 1992 cohort) 2 at screening, and 3) the mean value of two 75-g oral glucose tolerance tests (OGTT) in the IGT range based on WHO 1985 criteria The protocol for this trial and supporting CONSORT checklist are available as supporting information; see A total of 522 overweight men and women were randomly allocated to one of the two treatment modalities, the intensive diet-exercise counseling group  or the control group . The laboratory personnel were not aware of the group allocation but naturally the participants and staff members involved with the intervention were, so the study was only partly masked. The participants randomized to intensive lifestyle intervention were given individualized counseling by the study nutritionists to achieve the lifestyle goals. They were also advised to increase their level of physical activity, and voluntary physical activity sessions were offered. The lifestyle goals were 1) weight reduction of ≥5%, 2) <30% of the daily energy intake from fat, 3) <10% of the daily energy intake from saturated fat, 4) fiber intake ≥15 grams per 1000 kcal, and 5) moderately intense physical activity ≥30 minutes per day. The control participants were given general health behavior information at randomization All participants had an annual OGTT, a medical history, and a physical examination with measurements of height (without shoes), weight (in light indoor clothes), waist circumference (midway between the lowest rib and iliac crest) and systolic and diastolic blood pressure. Serum total cholesterol, HDL-cholesterol and triglycerides were determined from fasting samples using an enzymatic assay method.The FINRISK 1992 survey was carried out as a part of the FINMONICA cardiovascular risk factor survey, which is described in detail elsewhere The OGTT procedure and glucose tolerance classification was based on the WHO 1985 recommendations Individuals who reported that they have diabetes did not have an OGTT and were classified as known T2DM. Individuals with fasting plasma glucose level ≥7.8 mmol/l or 2 h plasma glucose ≥11.1 mmol/l were classified as having screen-detected type 2 diabetes (ST2DM). Those with 2 h plasma glucose ≥7.8 and <11.1 mmol/l, and fasting plasma glucose <7.8 mmol/l were classified as having IGT.Data on health status were collected through computerized register linkage to two nationwide health registers: the Hospital Discharge Register and the Causes of Death Register, using the national personal identification number.Data on all patients discharged dead or alive from all hospitals in Finland have been recorded in a computerized Hospital Discharge Register since year 1968. The diagnoses for hospitalizations have been coded according to the International Classification of Diseases (ICD) version 8 during 1967 to 1986, version 9 during 1987 to 1995, and version 10 since 1996.Both registers were complete with information until the end of year 2006. End points during follow-up were total mortality, and incident cardiovascular events , defined as the ICD 8 and 9 codes 401–449, and ICD 10 codes I10–I79. These codes include acute coronary events, coronary heart disease, stroke and hypertensive disease. In both registers the diagnoses are assigned by the physician treating the patient. Diagnostic classification in these registers has been validated against diagnoses derived by myocardial infarction registers, and the concordance was found high In order to estimate the baseline CVD risk according to the measured risk factors at baseline, we calculated for each study person their 10-year probability to have an CVD-event according to the Framingham risk equation The study protocols of both the DPS study and FINRISK survey were approved by the ethics committee of the National Public Health Institute in Helsinki, Finland. All study participants provided informed consent prior to the clinical assessments. In the DPS study, 17 participants did not give their consent for the register linkages, and they were thus excluded from this study.Participants were followed up until December 31, 2006, with a median follow-up time of 10.6 years for the DPS trial and 13.8 years for the FINRISK Study cohorts. We calculated mortality and CVD incidence rates separately for the DPS and FINRISK study cohorts, and by glucose tolerance status at the initial examination. In univariate analyses, log-rank test was used to assess differences between the glucose tolerance groups. In multivariate Cox models, the relationship between glucose tolerance at baseline and CVD during the follow-up was further analyzed, taking into account possible confounding factors including age, sex, smoking, history of CVD, systolic blood pressure and total cholesterol. All the analyses were done for the entire study cohorts and after exclusion persons who had had a CVD event before the first examination. Analyses were done with the statistics package Stata version 9.2 The DPS intervention and control groups were similar with regard to the main baseline variables . CompareCompared with the normoglycemic people in the FINRISK study, all the other groups (i.e. hyperglycemic people) had elevated triglyceride levels, and lower levels of HDL cholesterol .Among the DPS participants, 8.2% in the intervention group and 8.1% in the control group had CVD in the outset of the study. In the FINRISK study cohorts there was an increasing trend in the prevalence of CVD from 9.0% in the normoglycemic group to 34.8% in patients with known T2DM. Thus, the baseline prevalence of CVD in the DPS cohort was at the same level as in the FINRISK normoglycemic group.The estimated 10-year Framingham CVD risk in both of the DPS cohorts was similar to that in the FINRISK normoglycemic group.The total number of deaths during the follow-up were 214 (11%) in the FINRISK cohort, and 16 (3%) in the DPS cohort . Age- anIn the DPS cohort, after a median follow-up time of 10.2 years, there were 57/257 new CVD events in the intervention group and 54/248 in the control group . These cWe also examined CVD risk after excluding people with a history of CVD at baseline. The analyses showed essentially similar results as presented above for all participants, i.e. the highest CVD risk was found in people with T2DM. Further, in sensitivity analyses, we replicated all the analyses above by excluding early CVD-events, i.e. events occurring during the first 3 years after the baseline health examinations. The results remained essentially unchanged compared with the results presented here (data not shown).In the entire FINRISK cohort, excluding persons with history of CVD, baseline risk factors were strongly associated with CVD incidence . When adWhen all the variables in Finally, we analyzed the effects of changes in selected risk factors within the first year of the DPS study on the CVD risk . In unadOur main aim was to examine whether a lifestyle intervention with weight reduction, dietary counseling and increasing physical activity could reduce total mortality and CVD morbidity in the Finnish DPS. The CVD morbidity was identical for the DPS intervention and control groups. Thus, we found no evidence of overall CVD risk modification by the DPS lifestyle intervention in persons with IGT. In keeping with findings from numerous previous prospective studies Interestingly, total mortality in both groups of the DPS participants was markedly lower when compared with the FINRISK participants with IGT. The mortality data, however, should be interpreted with caution due to a small number of cases and as the difference could reflect the distinct selection procedures of the study populations, leading to divergent risk profiles as shown by differences in the risk factor levels and the calculated Framingham 10-year CVD-probability. However, besides these factors, also the regular follow-up and health consciousness of the DPS study participants could explain the low mortality among them.The incidence rates of T2DM in the DPS study during the overall follow-up of 7 years were 4.3 and 7.4 cases per 100 person-years in the intervention and control groups, respectively  The DPS was not initially planned to examine the effect of lifestyle intervention on total mortality or CVD morbidity. Therefore, the statistical power may not be sufficient to detect small differences in CVD events between the intervention and control groups in the DPS. Another explanation for the equal CVD risk in the lifestyle intervention and control groups in the DPS may be that lifestyle changes were not extensive enough to result in significant CVD risk reduction even though they were sufficient to reduce the rate of progression to T2DM. Nevertheless, in addition to the improvement in glycemia, some beneficial changes were achieved in other risk factors of CVD in the DPS As in many previous studies, low HDL-cholesterol and high triglycerides showed to be the main risk factors for CVD in both persons with IGT and T2DM In conclusion, the DPS lifestyle intervention that resulted in marked decrease in the incidence of T2DM even after the active intervention period did not decrease the risk of CVD during a 10-year follow-up. A lower initial CVD risk profile and regular follow-up may explain the observed lower mortality among the DPS participants when compared to the population based IGT or normoglycemic cohorts. In the future, lifestyle intervention studies to prevent T2DM should be focused on individuals, who besides an increased risk of T2DM also have a markedly increased CVD risk. The future studies should also be sufficiently powered to detect modest differences in CVD morbidity or mortality. Finally, meta-analyses of multiple intervention trials may offer an opportunity to solve the question on the effects of lifestyle changes on CVD morbidity and mortality among persons with IGT.Protocol S1Trial Protocol(0.05 MB DOC)Click here for additional data file.Checklist S1CONSORT checklist(0.06 MB DOC)Click here for additional data file.


Hairless mice that ingested arsenite in drinking water exhibited more than a 5-fold enhancement of ultraviolet radiation (UVR) carcinogenesis, whereas arsenite alone was carcinogenically inactive. Dietary organoselenium blocked the cancer enhancement effect of arsenic but not cancer induction by UVR.In this study we sought to explain selenium blockage of As enhancement by establishing the extent that As and Se tissue distributions are coincident or divergent.a) UVR, b) UVR + As, c) UVR + organoselenium, or d) UVR + As + organoselenium.We used the X-ray fluorescence microprobe at the Advanced Photon Source  to probe sections of skin and liver from hairless mice exposed to S-glutathionyl) arsinium ion in the liver.We found elevated levels of As in the skin epithelium (hair follicles and epidermis) and diffusely in the liver of mice exposed to UVR + As. Arsenic was entirely absent in skin in mice exposed to UVR + As + organoselenium, but a diffuse low level was seen in the liver. As and Se locations were consistently divergent in skin; As was more diffusely distributed, whereas Se was strongly associated with membranes. X-ray absorption near-edge spectra are consistent with the presence of the seleno-bis(S-glutathionyl) arsinium ion in the liver suggested that formation of this compound was more likely to be responsible for the As-blocking effect of Se than was a mechanism based on antioxidation.Supplemental Se was uncommonly effective at preventing even a trace of As in skin at 14 or 196 days of continuous exposure to As in drinking water. Traces of the seleno-bis( In the United States, more than 1 million new cases of nonmelanoma skin cancer are diagnosed each year. Ultraviolet A (UVA) radiation and sunlight exposure are well-established risk factors leading to squamous cell and basal cell carcinomas . UV radiParadoxically, arsenic is only weakly carcinogenic in the skin of laboratory animals . Recent Inflammation and its associated increase in ROS is frequently seen in skin exposed to UVR generally within 3–5 days after exposure . ROS cauIn mouse skin the antioxidants vitamin E and organoselenium blocked enhancement of UVR carcinogenesis by As , raisingS-glutathionyl) arsinium ion {[(GS)2AsSe]−} in the liver and bile, which is a possible alternative way for Se to interfere with the activity of As  microprobe at the Advanced Photon Source to track As and Se distributions in skin and liver.2 UVR was started three times weekly and continued until 196 days. The p-XSC was mixed into powdered rodent chow, and controls were fed the unsupplemented powder. The UVR source was a bank of four Westinghouse FS-20 fluorescent tubes  mounted in parallel 15 cm apart at a distance of 30 cm from the skin surface. The fluence rate, as measured by an erythemal radiometer , was 0.2 kJ/m2 per minute. Each individual UVR dose was approximately one-fourth of the mouse minimal erythemic dose, that is, 1.0 kJ/m2. The mice were treated humanely with regard to suffering in conformity to rules established by the Institutional Animal Care and Use Committee of the New York University School of Medicine.Hairless (HR-1) mice were obtained from Charles River Breeding Farms . Beginning at time 0 (28 days of age), mice were exposed to 5 mg/L As(III) in drinking water, to 10 ppm p-XSC in rodent chow, or to both compounds and continued for 196 days. At 14 days, a topical fluence of 1.0 kJ/mTissue samples were prepared for elemental analysis at 14 days and 196 days. Samples of skin and liver were excised, fast frozen, and sectioned on a cryotome. Sections were mounted on silicon nitride support windows and desiccated before attachment to kinematic sample holders mounted on a motorized x–y-stage of a Leica DMXRE epifluorescence microscope . Positional coordinates from selected locations were transferred to a corresponding x–y-stage for analysis of elemental distributions.We used XRF microprobe analysis to examine element distributions and concentrations in tissue sections at beamline 2-ID-E of the Advanced Photon Source. We used an Si(111) single-bounce monochromator to tune the undulator synchrotron X-ray beam to 12.9 keV. The beam was focused into a 0.5-μm spot using Fresnel zone plate optics. For element distribution maps, the sample was rastered through the focal spot with a step of 1–2 μm and dwell time of 4 sec. A full energy-dispersive XRF spectrum was collected at each step using a Canberra three-element UltraLEGe detector . Elemental maps were created by fitting the spectrum at each scan position with modified Gaussians using MAPS software . Area coWe obtained X-ray absorption near-edge spectra (XANES) at beamline 2-ID-D in fluorescence mode using a Si-drift detector. Spectra were obtained at As and Se K-edges for the liver sections only of the As + Se exposure because no As was found in other tissues. Multiple spectra were obtained (up to 17) and then averaged. Spectra were also acquired for sodium arsenite, disodium arsenate heptahydrate, sodium selenite, and sodium selenate as oxidation state references.The Advanced Photon Source provided opportunities to scan tissue sections to establish the distribution of metals and other elements and to make relatively long counts at specific loci for constructing comparative concentrations of the various elements. Several markers of membrane localization shown in Another view of the disassociation between the distributions of As and Se is shown in The spectra in 2AsSe]− and selenoglutathione [Se(GS)2] 2]  and in bSe(GS)2] . The disSe(GS)2] .2AsSe]− and arsiniumglutathione [As(GS)3] and suggests the presence of the same oxidation state for As in mouse liver as was seen in rabbit liver and bile  limits more detailed comparisons with published results. The As and Se concentrations used in the present study were chosen to correspond to levels at which carcinogenic enhancement has been observed in mice. Consequently, even though the observed spectra shown here are broadly consistent with those observed at much higher levels in rabbits, the results are not quantitatively sufficient for a definitive identification to be made. Nevertheless, the XANES results are consistent with the presence of [(GS)2AsSe]−, which is then eliminated from the body in the bile the bile . BlockinThe blockage afforded against As by Se was equally effective at 14 days of exposure as at 196 days, implying that the effectiveness of the inhibition occurs early and persists for long periods of time in the absence of noticeable toxicity by the Se. These data, coupled with the rapid achievement of equilibrium when the As is first started, suggest that the turnover time for As in this mouse model is relatively short, on the order of 1 or 2 days.p ≤ 0.05). The lack of an Se effect on UVR carcinogenesis is consistent with the finding that the Se is acting very differently than vitamin E . It also follows from the present results that little, if any, As was sequestered by the endogenous Se, presumably because most of the latter is normally locked inside of antioxidative and other enzymes  produced a 20% decrease in UVR carcinogenesis  raises a serious question whether human studies showing that As is a carcinogen by itself may have overlooked a low-level companion . PrecedeAlthough the mechanism of formation of As–Se complexes is not fully understood, current evidence indicates that exogenous As depletes glutathione pools and elevates arsenation of protein sulfhydryl groups . The levThe present study points to the use of dietary Se as a potentially effective countermeasure against any and all types of exposures to As in drinking water that may result in unhealthy outcomes, because the As is effectively sequestered and excreted in the presence of sufficient levels of Se.


Objective To validate use of the Manchester triage system in paediatric emergency care.Design Prospective observational study.Setting Emergency departments of a university hospital and a teaching hospital in the Netherlands, 2006-7.Participants 17 600 children (aged <16) visiting an emergency department over 13 months  and seven months .Intervention Nurses triaged 16 735/17 600 patients (95%) using a computerised Manchester triage system, which calculated urgency levels from the selection of discriminators embedded in flowcharts for presenting problems. Nurses over-ruled the urgency level in 1714 (10%) children, who were excluded from analysis. Complete data for the reference standard were unavailable in 1467 (9%) children leaving 13 554 patients for analysis.Main outcome measures Urgency according to the Manchester triage system compared with a predefined and independently assessed reference standard for five urgency levels. This reference standard was based on a combination of vital signs at presentation, potentially life threatening conditions, diagnostic resources, therapeutic interventions, and follow-up. Sensitivity, specificity, and likelihood ratios for high urgency (immediate and very urgent) and 95% confidence intervals for subgroups based on age, use of flowcharts, and discriminators.Results The Manchester urgency level agreed with the reference standard in 4582 of 13 554 (34%) children; 7311 (54%) were over-triaged and 1661 (12%) under-triaged. The likelihood ratio was 3.0  for high urgency and 0.5 (0.4 to 0.5) for low urgency; though the likelihood ratios were lower for those presenting with a medical problem (2.3 (2.2 to 2.5) v 12.0 (7.8 to 18.0) for trauma) and in younger children (2.4 (1.9 to 2.9) at 0-3 months v 5.4 (4.5 to 6.5) at 8-16 years).Conclusions The Manchester triage system has moderate validity in paediatric emergency care. It errs on the safe side, with much more over-triage than under-triage compared with an independent reference standard for urgency. Triage of patients with a medical problem or in younger children is particularly difficult. Emergency departments need systems to prioritise patients.2As “subjective” triage by nurses without using a system has low sensitivity and specificity, it is important to develop and evaluate triage systems.15We prospectively validated the Manchester triage system for children in paediatric emergency care. We conducted a large prospective study to allow for sufficient statistical power and detailed evaluation of specific categories of patients.In this prospective observational study we measured validity by comparing the assigned urgency categories of the Manchester triage system with a predefined independent reference classification of urgency. The study included children aged under 16 attending the emergency departments of two large inner city hospitals. The emergency department of the Erasmus MC-Sophia Children’s hospital (Rotterdam) is a university paediatric emergency department visited by about 9000 patients per year; the Manchester triage system has been in use here since August 2005. We included in our study children who attended from January 2006 to January 2007.The emergency department of the Haga Hospital-Juliana Children’s Hospital (The Hague) is a mixed paediatric-adult emergency department of a large teaching hospital visited by nearly 30 000 patients per year, of whom about half are children. The Manchester triage system was implemented at this site in 2003; we included children attending from January to July 2006.Both hospitals are in the southwest of the Netherlands, which has a population of about four million people and an annual birth rate of 47 000.16Emergency department nurses performed a short assessment and triaged patients using the Manchester triage system. The system is an algorithm based on flowcharts and consists of 52 flowchart diagrams (49 suitable for children) that are specific for the patient’s presenting problem. The flowcharts show six key discriminators  as well as specific discriminators relevant to the presenting problem. Selection of a discriminator indicates one of the five urgency categories, with a maximum waiting time . The presence of key discriminators in different flowcharts will lead to the same level of urgency. Pain is scored on a scale from 0-10 and could assign patients to a higher urgency level. If the nurse does not agree with the assigned urgency category, the system can be overruled. We used a computerised version that uses the official Dutch translation of the flowcharts and discriminators of the first edition (1996).17Patients’ characteristics, selected flowcharts, discriminators, and urgency category were recorded in the computerised triage system. Nurses or physicians recorded data concerning vital signs, diagnosis, diagnostic and therapeutic interventions, admission to hospital, and follow-up on structured electronic or paper emergency department forms. Trained medical students gathered and entered the data on a separate database, independent of the triage outcome, using SSPS data entry version 4. The database was checked for consistency and outliers. Data on laboratory tests were obtained from the hospital information system.Before the study we defined a reference standard based on literature and expert opinion.Patients were considered to be category 1 (immediate) if they had abnormal vital signs according to the paediatric risk of mortality score (PRISM).Patients were considered to be category 2 (very urgent) if their vital signs were within normal range and the presumed diagnosis at the end of their consultation in the emergency department was a potentially life threatening condition . Most of these conditions are associated with a high morbidity and mortality and are discussed in the advanced paediatric life support workbook as emergencies.24Patients were allocated to category 3 or 4 (urgent or standard) depending on the performed diagnostics, administered treatment, and the scheduled follow-up.Patients were considered to be category 5 (non-urgent) if they did not require any of the resources. Previous studies on other triage systems for children showed an association between urgency level and resource use and follow-up. Resource use is associated with the urgency level of the emergency severity index (ESI).A classification matrix of the reference classification and detailed definitions of the reference standard urgencies are shown in appendices 1 and 2 (see bmj.com). We defined the reference standard for each patient independent of urgency according to the Manchester system and based on a computerised application of the classification matrix.In our pilot study 1% of the patients were classified as immediate.We validated the Manchester triage system by comparing the assigned urgency category with the category assigned with the reference standard. We defined over-triage and under-triage as the proportions of patients who had a higher or lower urgency category with the Manchester system, respectively, than with the reference standard.15http://statline.cbs.nl/statweb).16We calculated sensitivity, specificity, and likelihood ratios for classification as high urgency and low urgency (likelihood ratio+=sensitivity/(1−specificity) and likelihood ratio−=(1−sensitivity)/specificity).Nurses applied the Manchester triage system in 16 735 of 17 600 children (95%) who attended the emergency department. The distribution of the reference standard did not differ between those who were or were not triaged (P=0.06). Nurses over-ruled the urgency category in 1714 (10%); 735 of whom (43%) had originally been triaged with the Manchester triage system as very urgent compared with 21% of the patients triaged with the Manchester system overall. Of these children in whom the classification of very urgent was over-ruled, 720 (98%) were downgraded by at least one category.In the 384 and 509 patients triaged into the urgent and standard categories of the Manchester triage system, 73 (19%) and 22 (4.4%), respectively, were downgraded by at least one category. Fever discriminators (27%) and the discriminator of recent problem (22%) were often used if the urgency category was overruled.In 1467 (9%) children, complete data were unavailable for the reference standard, leaving 13 554 for analysis. Distribution of the urgency category among children in whom the reference standard was missing was comparable with that in those without missing data (P=0.14). Median age was 3.4 years (interquartile range 1.2-8.0), 6631 (49%) children attended the university hospital, 5740 (42%) were female, and 6965 (51%) were not referred by a general practitioner or medical specialist. v 277 (2%)). Considerably fewer children were classified as non-urgent with the Manchester system than with the reference standard (112 (1%) v 2927 (22%)) (fig 1)Classification of urgency according to the Manchester triage system and the reference standard agreed in 4582 (34%) children. More children were classified as very urgent with the Manchester system than with the reference standard (2897 (21%) The Manchester urgency level agreed with the reference standard in 34% (n=4582). Some 5001 (37%) children were over-triaged by one category and 2310 (17%) by more than one category. With the Manchester system 1474 (11%) were under-triaged by one category and 187 (1%) by more than one category. Agreement with the reference standard was particularly low for the very urgent category, with only 119 of 2897 (4%) classified correctly; 2545 (88%) were over-triaged and 233 (8%) patients were under-triaged (fig 1).The validity of the Manchester system in children triaged with medical flowcharts differed considerably between the top 10 medical flowcharts, with poor validity for the worried parent flowchart  (fig 2 and table 1)Commonly used general discriminators were recent problem (20%), pain discriminators (17%), fever discriminators (15%), recent injury (9%); commonly used specific discriminators were increased work of breathing (4%) and persistent vomiting (4%). Patients triaged with a fever discriminator showed a low validity, especially with increasing age (fig 3).The Manchester triage system has an overall moderate validity compared with an independent reference standard. The agreement with the reference standard was 34%, with over-triage in 54% and under-triage in 12% (mostly by one category). The sensitivity for high urgency was 63%, implying that 37% of the patients who actually needed to be seen within 10 minutes were not categorised as that urgent. The specificity was 79%, implying that 21% low urgency patients were categorised too high. In particular, patients in the very urgent category were over-triaged.The validity was lower in children presenting with medical problems compared with those presenting with trauma. Any modifications should therefore be particularly targeted for medical problems. Specific discriminators can be considered for their role in the triage system. For example, children aged <3 months with fever are at greater risk for a serious bacterial infection, whereas children aged ≥3 months with fever might be allocated to a lower urgency category.29The validity of triage systems depends on the extent to which the system predicts urgency and on the accuracy of the nurse who applies the system (inter-rater agreement). We previously found a good inter-rater agreement of the Manchester system in children at our two emergency departments, both for written case scenarios  and for simultaneous triage of actual patients  . We can therefore assume that the validity of the Manchester system compared with the reference standard is mostly due to the predictive value of the system to assess urgency.In the Manchester triage system, conditions such as shock, inadequate breathing, compromised airway, and unresponsiveness are used to identify children who need to be seen immediately. For the reference standard we classified children as immediate if blood pressure, heart rate, and respiratory rate were abnormal, if they had a decreased consciousness, or if hyperpyrexia or hypothermia was present. As abnormal vital signs predict mortality in children in critical care unitsFurthermore, the reference standard is based on a combination of patients’ characteristics collected at the time of presentation and at the end of the consultation in the emergency department. Characteristics gathered at the end of the consultation might be less suitable to define urgency because of possible changes in the patient’s condition over time. Assessment of true acuity requires more information than is available at the time of triage.Our reference standard can therefore be seen only as an approximation of an ideal standard as it was previously used to study the Manchester triage system in paediatric emergency care and had the advantage of classifying patients across five urgency categories.33Another limitation of the study is that nurses over-ruled the Manchester system urgency category in 10% of the patients. Originally, these patients were often allocated to the very urgent category, which showed a low validity. Inclusion of the 10% over-ruled patients would probably have lowered the validity of the Manchester system.Furthermore, data for the reference standard were missing in 9% of the patients. Selection bias is not likely as the distribution of the Manchester system categories for patients with missing data was similar to that of the patients without missing data.Finally, the study was performed in a large urban mixed paediatric-adult emergency department and a large university paediatric emergency department with 90% basic paediatric care. Although these two centres might have a relatively larger number of immediate cases, they are likely to be representative of large emergency departments.In an earlier retrospective evaluation of the Manchester triage system in paediatric emergency care, we found 40% correctly triaged, 15% under-triaged, and 45% over-triaged.Other triage systems studied in paediatric emergency care show a high validity (Soterion rapid triage system),The use of an independent reference standard for each patient will allow for further development and evaluation of modifications to the Manchester triage system. When applying the Manchester triage system in paediatric emergency care, users should be aware of its moderate validity. We need to consider and study modifications for specific flowcharts, discriminators, and age groups for which the triage system has a low validity.The Manchester triage system shows a moderate validity in paediatric emergency care but errs on the safe side as the percentage over-triage is much larger than under-triage compared with a reference standard for urgency. Triage of patients with a medical problem or younger patients is particularly difficult. The consensus based five level Manchester triage system is sensitive in identifying seriously ill adults and those with high risk chest painAlthough the system is widely applied, a large prospective study to evaluate the validity on its use in children is lackingIn paediatric emergency care the Manchester triage system shows moderate validity but errs on the safe side as the proportion of over-triage is much larger than under-triageTriage of children with a medical problem or young patients (aged <1 year) was particularly difficult and the system should be specifically modified to cope with such cases


Genes with short 5'UTR introns have higher expression than genes with no or long 5'UTR introns. Complex evolutionary forces act on these introns. Approximately 35% of human genes contain introns within the 5' untranslated region (UTR). Introns in 5'UTRs differ from those in coding regions and 3'UTRs with respect to nucleotide composition, length distribution and density. Despite their presumed impact on gene regulation, the evolution and possible functions of 5'UTR introns remain largely unexplored.We performed a genome-scale computational analysis of 5'UTR introns in humans. We discovered that the most highly expressed genes tended to have short 5'UTR introns rather than having long 5'UTR introns or lacking 5'UTR introns entirely. Although we found no correlation in 5'UTR intron presence or length with variance in expression across tissues, which might have indicated a broad role in expression-regulation, we observed an uneven distribution of 5'UTR introns amongst genes in specific functional categories. In particular, genes with regulatory roles were surprisingly enriched in having 5'UTR introns. Finally, we analyzed the evolution of 5'UTR introns in non-receptor protein tyrosine kinases (NRTK), and identified a conserved DNA motif enriched within the 5'UTR introns of human NRTKs.Our results suggest that human 5'UTR introns enhance the expression of some genes in a length-dependent manner. While many 5'UTR introns are likely to be evolving neutrally, their relationship with gene expression and overrepresentation among regulatory genes, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns. The advent, evolution and functional significance of introns in eukaryotes have been topics of intense debate over the past 30 years . TherNeutral or nearly neutral population genetic processes under general, non-adaptive conditions have been suggested to result in dynamic gains and losses of introns. Such neutral processes could account for some of the observed patterns of intron presence , but do Most of the theoretical and empirical work on the evolution of introns has focused on those found in coding regions, yet an appreciable fraction of human genes (approximately 35%) contain introns in their 5'UTRs . IntronsCaenorhabditis elegans [Oryza and Arabidopsis, such that highly expressed genes have more and longer introns [As 5'UTR introns (5UIs) are unusually long and can considerably increase the total number of bases transcribed for a given gene, it is useful to consider the two main adaptationist theories about the functional consequences of intron length. The first model argues that it is energetically costly for cells to transcribe long stretches of DNA that does not encode protein . By this elegans , and the elegans ,25. Howe introns . If the The second model, termed 'genome design', posits that the pressure to maintain many intronic regulatory elements favors longer introns in tissue-specific genes . The maiCryptococcus, an intron-rich fungus with longer 5' and 3' UTR introns than coding region introns [Neither of these two principal theories addresses the possible role of 5UIs and the evolutionary pressures acting on them; therefore, the functional significance, if any, of their frequent occurrence remains unclear. Given that splicing of these sequences seemingly has no effect on the amino acid sequence of the encoded protein, it is unclear what selective benefit might accompany their removal from the mature mRNA. The reduced splice-site conservation and high variability in length of 5UIs have led to the suggestion that they contract and expand without significant functional consequences . However introns  and high introns .Given these conflicting results and the scarcity of studies regarding the evolution of UTR introns, it is worthwhile to consider a functional perspective. An analysis of functional trends among human genes with 5UIs could lead to a better understanding of their evolution and also potentially to the detection of novel mechanisms of regulation mediated by these introns. Here, we analyze expression profiles of genes with 5UIs and examine the distribution of these introns in different functional categories of genes.To investigate the functional properties of human 5UIs, we used NCBI's Reference Sequence (RefSeq) collection. These are curated, full-length sequences with annotated UTR boundaries, and expression data are available for many of them. The lack of a translation reading frame makes the computational prediction of splice sites in 5'UTRs inherently more difficult , necessiP < 2.2e-16). In fact, when genes with 5UI lengths in the lowest 25th percentile were analyzed, the correlation was no longer significant . A statistically significant, albeit slight, correlation was found for genes with 5UI length below the median . Among the genes with 5UIs, a similar relationship was evident between the total length of 5UIs and the total length of the remaining introns , the relationship was clearly driven by the genes with longer 5UIs. When genes with 5UI lengths either in the lowest 25th or 50th percentile were considered, correlation was negligible .We next considered the relationship between the total lengths of 5'UTR exons and of 5UIs. Even though there was a correlation between the lengths of 5UIs and 5'UTR exons overall, this correlation was slight and was driven by the genes with the longest 5UIs  and also in the top 5%   Figure . These gP = 1.53e-11 and P = 3.21e-10, respectively).The enrichment for high expression in genes with short 5UIs held even when genes with the longest 25% of 5UIs were removed. In this case, the genes with the highest 1% and 5% expression were, respectively, 9.5 times and 2.5 times more likely to have short 5UIs as opposed to intermediate length 5UIs ; and the remaining genes. For the most highly expressed genes, the inter-quartile range of total 5UI length was 215 to 734 nucleotides compared with 289 to 870 nucleotides for the remaining genes Figure . Thus, tP = 0.25). Also, no statistically significant depletion for the long 5UI category was observed in either the top 1% or the top 5% expression group . Thus, we did not observe the inverse relationship between expression and total 5UI length that might have been expected under the energetic cost model.Interestingly, no expression dependence was observed among genes with intermediate or long 5UIs: genes with long 5UIs (top 25th percentile in length) did not tend to be expressed less than those with the intermediate length 5UIs . However, genes with short 5UIs were 1.8 times more likely to be in the top 5% and 3.3 times more likely to be in the top 1% in overall expression level than genes with no 5UIs  than genes with no 5UIs . Therefore, the tendency of highly expressed genes to have short 5UIs is unlikely to be confounded by the effects of 5'UTR exons or the remaining introns.The observed expression trends could reflect the influence of genomic features other than 5UIs. Yet, short 5UIs do not seem to predict a short total length of either non-5'UTR introns or 5'UTR exons Figure . FurtherFor genes with the highest expression levels, these results are in contrast to the neutral model of 5UI evolution, which predicts that 5'UTR intronic length should not depend on expression level. These results are also not explained by the energetic cost hypothesis, which would predict that genes with the highest expression levels should be less likely to have 5UIs. In stark contrast to the predictions of each model, we found the most highly expressed genes to be significantly enriched in short 5UIs. Furthermore, the energetic cost hypothesis would also predict a linear decrease in the total 5UI length as a function of increasing gene expression. Yet, we found no overall differences with respect to 5UI length except for the most highly expressed genes. Even though a neutral model of 5UI evolution is plausible for most genes, our results for the most highly expressed genes are inconsistent with both neutral and energetic cost models Figure .We next used expression to assess the applicability to 5UIs of the other major hypothesis of intron evolution, the 'genome design model', which predicts that intermediate or long introns should be enriched in tissue-specific genes as a consequence of complex regulation. As originally outlined, the genome design model explicitly disregards 5UIs ; howeverWe sought to address two potential sources of bias. First, gene expression levels vary greatly and variance is strongly correlated with mean expression. Therefore, we calculated the standard deviation-to-mean ratio (coefficient of variation or CV) , a normaCVx is the CV of expression of gene x across all tissues, yx represents the vector of CV values for all 201 genes in a window centered around gene x, while μ1/2 and MAD represent the median and median absolute deviation, respectively. As expected, genes with low expression tended to have much more variability across tissues . We then examined the lengths of the introns as a function of variability in expression , but the genes with highest 1% across-tissue variability tended to have slightly shorter 5UIs . Genes with short 5UIs were also overrepresented in the top 1% across-tissue variability category . Our results suggested that length of the 5UI was not a major factor in determining across-tissue variability but there was a preference for shorter 5UIs in the most variable genes.where s Figure . Based os Figure . The remn Figure . The genP = 0.19). Furthermore, the distribution of number of tissues in which each gene was present did not differ between genes containing and lacking 5UIs  function annotations to determine which groups of genes are enriched or depleted in 5UIs, if any. Two popular functional trend analysis tools, FuncAssociate  and GoStSRC, c-src tyrosine kinase (CSK), janus kinases (JAK), spleen tyrosine kinase (SYK), tec protein tyrosine kinase (TEC), and Bruton agammaglobulinemia tyrosine kinase (BTK) among others.A significant overrepresentation of genes with 5UIs was found in many regulatory pathways Table . Non-recTo gain insight into the evolution of NRTK 5UIs, we identified orthologous genes in mouse and rat genomes corresponding to each human NRTK. We collected 5'UTR features for these genes in each genome using RefSeq annotations . Another current limitation is ambiguity in identifying the specific branch in which a given deletion or insertion event took place. Despite these shortcomings, a comparison of orthologs already provides insight into the dynamics of the evolution of 5UIs in NRTK genes.UTRs are also generally less well defined in less intensively studied organisms. For example, P < 0.0001 for all; Figure P = 1.4e-07).When every ortholog of a given NRTK had at least one annotated 5UI, the lengths of those introns were generally highly correlated Figure . Given tFES changed by less than five nucleotides in all possible comparisons, rat PTK2 and mouse PTK2 5UIs differed by approximately 63.5 kb . Given that the median position of the first 5UI was approximately 130 nucleotides away from the transcription start site regardless of the number of 5UIs , we defiWe next used GO annotations to compare the functional properties of 5UI-lacking genes with 5PCIs to those without 5PCIs. We observed the strongest enrichment of 5PCIs among genes in the following functional groups: MHC protein complex 1, cytosolic ribosome, hemoglobin complex, glutathione transferase activity, and transmembrane transporters , but 5UI presence or absence alone (without considering 5UI length) does not correlate with gene expression. Therefore, for 5UIs, short intron length seems to be a more important predictor of a high expression level than the presence or absence of 5UIs.rubi3 [Given the inconsistency between our observations and the energetic cost hypothesis, we suggest two alternative models of 5UIs' effect on gene expression. The first model is that splicing-dependent enhancement in gene expression is influenced not only by the position of an intron, but also its size. The second model is that transcriptional regulatory proteins are recruited as a result of the presence of DNA elements, which in turn enhance expression level. This process could be restricted spatially, such that if the distance between the regulatory element and the transcription start site is long, then the enhancement should be less pronounced. Hence the genes with the highest expression levels might be under selective pressure to keep their introns short in order to retain their enhancer elements closer to the transcription start site. In this scenario, one can further imagine these elements to function in a tissue-specific regulatory mechanism if the recruited factors are themselves tissue-specific. Such an enhancer, located in the first intron of the mammalian acetylcholinesterase gene, was previously found to mediate the tissue-specific expression of this gene . Anotherrubi3 .cis-regulatory modules could be contained within even a short (<1 kb) 5UI. Therefore, 5UIs need not be particularly long to enable complex and conserved regulation via cis-regulatory elements. Our results support the idea that the genome design model is not likely to be the most useful guide for understanding the evolved lengths of 5UIs.The pressure to maintain regulatory elements in introns is also the central idea of the genome design model, and we tested the applicability of this hypothesis to 5UIs by analyzing genes with tissue-dependent variability in gene expression. As the most proximal intron to the transcription start site has been shown to contain more regulatory elements ,34, the Finally, we considered whether certain classes of genes preferentially include 5UIs, and whether 5UIs contain regulatory elements. We found that genes with regulatory functions are enriched for 5UIs. The non-receptor tyrosine kinases, which play fundamental roles in all aspects of cell biology and signal transduction, were the most strongly enriched gene category. We identified a conserved DNA motif in the 5UIs of many non-receptor tyrosine kinases that could function by recruiting transcription factors. This recruitment might lead to tissue- or condition-specific regulation of NRTKs. For example, in the gene encoding Bruton's tyrosine kinase (a non-receptor tyrosine kinase), an SP1 transcription factor binding site was identified within the 5UI . FurtherIt is worth considering other forms of selection pressure that might affect 5'UTRs and therefore 5UIs. Upstream AUGs (uAUGs) tend to decrease translational efficiency, so that highly expressed genes should tend to avoid uAUGs in exons. On the other hand, intronic uAUGs are spliced out before the mature message encounters the cytoplasmic translation machinery; hence, they should not have a similar effect. The negative selection pressure against exonic uAUGs that tends to favor increased intronic sequence content within 5'UTRs  should bBRCA1 and ERβ was recently implicated in carcinogenesis [Alternative splicing has emerged as a fundamental mechanism of regulation and expansion of the proteome, with nearly 95% of all genes thought to be alternatively spliced in mammals -15. Tissogenesis . WhetherNOD2, which is associated with Crohn's disease. Only a subset of NOD2's multiple splice variants include the uAUGs in the mature mRNA, and these have decreased translation efficiency [ELK1, for example, a facultative secondary structure modulates translation initiation [The functional importance of alternative splicing in 5'UTRs is exemplified by human ficiency . Alternaitiation . Yet anoitiation . The posOur results highlight the functional importance of 5'UTR introns. Existing models predicting selective effects, such as avoidance of uAUGs, minimization of transcriptional cost, or accumulation of regulatory elements, do not suffice to explain results from our genome-scale analysis of 5UIs. Given 5UI enrichment and depletion in specific functional categories of genes, and the potential ability of 5UIs to enhance gene expression, a complex interplay of multiple selective forces appears to have influenced the evolution of this distinct class of introns.NCBI's human Reference Gene Collection (RefSeq)  and the The microarray data were downloaded from Gene Expression Atlas, which included expression data from 79 different tissues in humans . We used10 odds ratio. All reported log odds ratios were obtained from FuncAssociate. Similar results were obtained using GoSTAT (data not shown).GoSTAT  and FuncTo study the evolution of 5UI presence and length among NRTKs, we first identified orthologs of human NRTKs in the mouse and rat genomes. We used NCBI's Homologene Release 64   overlapped by more than 20 base pairs. Then, we randomly removed RefSeqs transcripts such that only a single representative transcript remained for each equivalence class. Third, exact duplicates with respect to the 5'UTR were removed. Specifically, if two or more RefSeq IDs had the exact same 5'UTR, a single identifier was selected as a representative for that particular region. Splice variants that differ in their 5'UTR were not removed because these provide additional information about the lengths of 5'UTR introns and exons. All three methods yielded similar results and led to identical conclusions. Therefore, only one representative method is shown in the figures. The third method conveys the most information when discussing total 5UI lengths and hence was used in Figure For the specific GO categories used in our analysis, all the genes in a given category were retrieved from the human GOA database . The cor5PCI: 5' proximal coding region intron; 5UI: 5'UTR intron; CV: coefficient of variation; EST: expressed sequence tag; GO: Gene Ontology; kb: kilobase; NRTK: non-receptor protein tyrosine kinase; PCC: Pearson Correlation Coefficient; uAUG: upstream AUG; UTR: untranslated region.CC carried out all analyses, designed the study and drafted the manuscript. AD contributed to the generation of the 5UI dataset, provided guidance with all the analyses and contributed to the writing of the manuscript. JCM participated in the design of the study. GFB helped with functional enrichment analysis and contributed to the writing of the manuscript. FPR conceived and supervised the study, and contributed to the writing of the manuscript. All authors read and approved the final manuscript.Complete list of RefSeq mRNA IDs that have 5'UTR introns. This file contains the genomic coordinates and RefSeq IDs for all transcripts with 5'UTR introns. '+'and '-' represent the forward and reverse strands, respectively.Click here for fileComplete list of 5'UTR intron lengths for the human non-receptor tyrosine kinases and their orthologs in mouse and rat genomes. This file contains the RefSeq IDs and gene symbols for all human NRTKs and their mouse and rat orthologs. For all transcripts, 5'UTR intron lengths are given.Click here for fileOverrepresented GO attributes for genes with 5'-proximal coding introns. This file contains the table of overrepresented GO attributes for genes with 5'-proximal coding introns. The methods and legend are the same as in Table Click here for file


Despite evidence of the benefits of research use in post-acute stroke rehabilitation where compliance with clinical practice guidelines has been associated with functional recovery and patient satisfaction, the rate of reliance on the research literature in clinical decision making among physical therapists is low. More research examining factors that motivate physical therapists to consider research findings in neurological practice is needed to inform efforts to intervene. The objective of this study was to identify practitioner, organizational, and research characteristics associated with research use among physical therapists providing services post-stroke.A cross-sectional mail survey of physical therapists providing services to people with stroke in Ontario, Canada was conducted. The survey questionnaire contained items to evaluate practitioner and organizational characteristics and perceptions of research considered to influence evidence-based practice (EBP), as well as the frequency of using research evidence in clinical decision making in a typical month. Ordinal regression was used to identify factors associated with research use.The percentage of respondents reporting research use in clinical decision making 0 to 1, 2 to 5, or 6+ times in a typical month was 33.8%, 52.9%, and 13.3%, respectively (n = 263). Academic preparation in the principles of EBP, research participation, service as a clinical instructor, self-efficacy to implement EBP, a positive attitude towards research, perceived organizational support of research use, and Internet access to bibliographic databases at work were each associated with research use and placed in the final regression model. In the final model (n = 244), academic preparation in EBP, EBP self-efficacy, agreement that research findings are useful, and research participation each remained significantly associated with research use after adjusting for the effects of the other variables in the model.A third of therapists rarely use research evidence in clinical decision making. Education in the principles of EBP, EBP self-efficacy, a positive attitude towards research, and involvement in research at work may promote research use in neurological physical therapy practice. Future research is needed to confirm these findings and to determine the type of research participation that may promote research use. Evidence-based medicine has been described as 'the conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients' . NumerouDespite the expected benefits of implementing EBP, a repeated finding of qualitative and survey research is that physical therapists do not readily consult the research literature to inform clinical decision making -9. Few sTo narrow the knowledge to practice gap, we need to identify factors that exert the greatest influence on research use. The limited research that has been conducted to date has shown that although a high level of training is associated with research use among physical therapists , the numThe existing literature highlights specific practitioner characteristics -- such as insufficient education in the principles of EBP, and skill and self-efficacy to search, appraise, and apply findings from the research literature in clinical practice -- as substantial barriers to EBP ,16-18, pi.e., a written requirement) supporting EBP [Factors related to the healthcare organization that may restrict physical therapists' engagement in EBP include lack of a mandate  and use of the research literature in clinical decision making . Ordinal regression is used when the number of response categories for the dependent variable exceeds two [Ordinal logistic regression  was usedeeds two .Independent variables were re-categorized to form binary variables in the following manner before performing ordinal regression. For items with positively worded statements, we pooled the 'strongly agree' and the 'agree' categories to form an 'agree' category and collapsed the 'neutral,' 'disagree' and 'strongly disagree' categories to form a 'disagree' category. For items with negatively worded statements, we combined the 'strongly disagree' and the 'disagree' categories to form a 'disagree' category and pooled the 'neutral,' 'agree' and 'strongly agree' categories to form an 'agree' category. For items rated as 'yes,' 'no,' or 'do not know,' we combined the 'no' and 'do not know' categories assuming that not knowing about the presence of a resource, for example, would be similar to not having the resource .To ensure reliable estimates of association, we pooled categories of demographic variables with low cell counts . AlthougWe conducted ordinal instead of logistic regression to minimize information lost in using a two-category outcome . OrdinalIn ordinal regression, the c-statistic is used as a measure of the discriminative power of the model . A valuei.e., 2 to 5 times versus 0 to 1 time, 6+ times versus 0 to 1 time and 6+ times versus 2 to 5 times). If a variable was significantly associated with research use within each block  excluded 1) for at least one of the three comparisons, we included it in the final multivariable model and reported ORs and associated 95% CIs [The approach to analysis was exploratory given the paucity of research in this area. We took a blocked modeling approach and first entered subgroups of independent variables into separate models with research use as the dependent variable . For exa 95% CIs . We usedWe mailed the questionnaire to 1,155 physical therapists and, of these mailings, 43 envelopes (3.7%) were returned to sender. Of the remaining 1,112 mailings, 702 individuals (63.1%) returned a questionnaire. Among these responders, 334 (47.6%) were eligible to participate in the study. Of the eligible respondents, 270 (80.8%) returned a completed questionnaire and 64 people (19.2%) refused to participate. We analyzed data from the final sample of 270 physical therapists.We describe respondents and their practice settings in Table The percentage of respondents who reported using the research literature in clinical decision making at different frequencies in a typical month (n = 263) was: 0 to 1 time (33.8%), 2 to 5 times (52.9%), 6 to 10 times (6.8%), 11 to 15 times (2.7%), 16+ times (3.8%).Factors that were associated with research use in each block of independent variables included academic preparation in EBP, EBP self-efficacy, disagreement that there is a divide between research and clinical practice, agreement that literature and research findings are useful in daily practice, serving as a clinical instructor, research participation, perceived facility support of research use, and Internet access to bibliographic databases at work. Table The final multivariable model was based on a complete dataset from 244 participants due to missing data on select questionnaire items. Academic preparation in EBP, EBP self-efficacy, agreement that research findings are useful and research participation each remained significantly associated with research use in clinical decision making after adjusting for the effects of the other variables in the model  and EBP self-efficacy  were associated with research use 2 to 5 times versus 0 to 1 time a month. Physical therapists who perceived research as useful in daily practice were 2.7 times more likely than those who did not perceive research as useful to use research 2 to 5 times compared to 0 to 1 time in a month after adjusting for the remaining variables in the model. Physical therapists with high ratings of EBP self-efficacy were 2.2 times more likely than peers who rated their self-efficacy 20% lower to use research literature 2 to 5 times in a typical month compared to 0 to 1 time  after adjusting for the remaining variables in the model.Physical therapists who agreed that research findings are useful were 14.2 times more likely than those who did not agree to use research literature 6+ times versus 0 to 1 time a month after adjusting for the remaining variables in the model . Therapists who reported participating in research were 4.3 times more likely than those who did not participate to use research literature 6+ times versus 0 to 1 time a month  after adjusting for the remaining variables in the model.Factors that were associated with research use 6+ compared to 2 to 5 times included academic preparation in EBP  and research participation . Physical therapists with academic preparation in EBP were 4.1 times more likely than those without to use research in clinical decision making 6+ compared to 2 to 5 times a month. Further, therapists who participated in research were 2.8 times more likely compared to those who did not participate to use research 6+ compared to 2 to 5 times a month.In this exploratory study, we observed a moderate rate of research use in clinical decision making and identified a number of factors associated with research use among Canadian physical therapists providing healthcare services to people with stroke. Results indicate that a third of physical therapists use research findings in clinical decision making rarely or not at all, and only 13.3% of therapists integrate research in clinical decision making 6+ times in a typical month. The actual rate of research use in clinical decision making may be lower than that observed in this study for two reasons: participants may be more engaged in EBP activity than non-responders and some may have wished to provide socially desirable answers.The frequency of research use that we observed, however, was slightly lower than corresponding rates reported following a survey of American physical therapists where approximately 25% of therapists used research 0 to 1 time and 26% used research 6+ times in a typical month. Sampling physical therapist members of the American Physical Therapy Association, as well as the inclusion of participants with doctoral-level training in the American study, may explain differences between study results, particularly given our finding that education in EBP is associated with self-reported research use.i.e., mastery experiences) are likely to boost students' self-efficacy and performance of these activities following graduation. Postgraduate education targeting competence in EBP that provide opportunities for applying skills to current patient scenarios in the clinical practice environment may be more likely than interventions carried out external to the practice environment to elicit a change in subsequent clinical behavior [It is important to understand how the correlates of research use identified in the current study complement our previous analysis of antecedent behaviors such as searching and reading the research literature , and invbehavior  due to tbehavior .Although we measured self-efficacy of individual practitioners in the current study, related constructs that are particularly relevant to effective functioning of interdisciplinary stroke teams in implementing EBP include collective efficacy , the shaAttitude is a construct closely related to self-efficacy beliefs, and our findings show that attitudes can help to explain the implementation of EBP. Although we modeled therapists' responses to a number of statements evaluating attitudes towards EBP, such as EBP 'is necessary,' 'improves the quality of care,' and 'helps with decision making,' only the perceived usefulness of research findings in daily practice corresponded to therapists' degree of research use in clinical decision making. This particular attitude discriminated both moderate- and high-level users of research from low-level users who reported using research none or one time in a typical month in addition to relating to frequency of reading the research literature . Entry-lParticipation in research activities for some percentage of work time seems particularly important as it has been associated with searching, reading , and, inFinally, the significant association observed between perceived facility support of research use and therapists' research use in clinical decision making deserves note. Perceived facility support was independently associated with three EBP behaviors: searching, reading , and, ini.e., academic preparation, attitude towards research, self-efficacy, research participation, being a clinical instructor,) and the organization , underscoring that efforts to promote evidence-based physical therapy practice cannot target the physical therapist alone.Overall, this study revealed significant and modifiable correlates of research use at the level of the practitioner (The cross-sectional study design limits inferences that the associations observed are causal in nature. Findings may be limited to the Canadian context where the highest entry-level professional physical therapy programs are at the Masters level. The strengths of this research are the use of conceptual and behavior change frameworks to identify potentially influential variables related to research use. Participants were sampled from the provincial registry of physical therapists and not from membership lists of professional associations, which supports the generalizability of results to physical therapists in neurological practice. Finally, the use of a three-category dependent variable in statistical modeling and comparison of pairs of dependent variable categories optimized identification of variables most closely linked to specific levels of research use.Although a third of physical therapists rarely use research evidence in clinical decision making, results suggest a number of potential mechanisms through which to improve research use among physical therapists providing services to people with stroke. Future investigations of EBP should focus on the effects of academic training, self-efficacy beliefs, perceptions of the usefulness of research evidence in daily clinical practice, participation in research activities at work, supervision of trainees, organizational support of research use, and provision of Internet access to bibliographic databases at work.The authors declare that they have no competing interests.NMS conceived of, designed, and carried out the study, planned the statistical analysis, and drafted the manuscript. SJTG helped plan the analysis, performed the statistical analysis, and assisted in drafting the manuscript. SBJ and DAD contributed to conceiving the study. All authors read, provided critical input on and approved the final manuscript.


Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets.CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment. CpGlobal utilizes methyl-sensitive restriction enzymes, HRP Neutravidin to detect the biotinylated nucleotides incorporated in an end-fill reaction and a luminometer to measure the chemiluminescence. The assay shows high accuracy and reproducibility in measuring global DNA methylation. Furthermore, CpGlobal correlates significantly with High Performance Capillary Electrophoresis (HPCE), a gold standard technology. We have applied the technology to understand the role of global DNA methylation in the natural history of lung cancer. World-wide, it is the leading cause of death attributed to any cancer. The survival rate is 15% over 5 years due to the lack of any clinical symptoms until the disease has progressed to a stage where cure is limited.We have designed and developed an assay, Through the use of cell lines and paired normal/tumor samples from patients with non-small cell lung cancer (NSCLC) we show that global DNA hypomethylation is highly associated with the progression of the tumor. In addition, the results provide the first indication that the normal part of the lung from a cancer patient has already experienced a loss of methylation compared to a normal individual.CpGlobal may have a role as a barometer for the onset and development of lung cancer.By detecting these changes in global DNA methylation,  The functional role of DNA methylation includes maintaining the stability of chromosomes, silencing repetitive sequences, arresting the deleterious effects of integrated foreign DNA and controlling gene expression . Genome-CpGlobal, a non-radioactive, non-PCR, high-throughput approach to measure global DNA methylation. It describes how CpGlobal is performed in a microtiter plate, which enables multiple samples to be analyzed simultaneously. Furthermore, this paper illustrates how the assay utilizes biotinylated nucleotides to provide a highly accurate and reproducible method that can measure global DNA methylation using only 100 ng of genomic DNA per reaction. In addition, we demonstrate that CpGlobal is an excellent alternative to HPCE, one of the gold standard technologies.Several technologies have been developed to measure the methyl content of the genome. In general the techniques have been focused around the use of methodologies that can quantitate the 5-methyl cytosines using reversed-phase high performance liquid chromatography (RP-HPLC), two dimensional thin layer chromatography (2D-TLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), high performance capillary electrophoresis (HPCE) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) . These aCpGlobal has been employed to study the role of global DNA methylation in lung cancer to understand further the biology of this disease. It is the world's most common fatal cancer with an overall survival rate of 15% over 5 years [ 5 years . This di 5 years . In orde® chemiluminescence substrate  was added to each well. After 2 minutes the luminescence emitted from each well was quantitated by a Wallac Envision 2100 multilabel reader .To quantitate the amount of DNA methylation in any genome 100 ng of a sample was aliquoted nine times into a 96 well white Microfluor 2 plate . The genomic DNA in the first three wells was digested with 5 units of a methyl-sensitive restriction enzyme such as HpaII , the DNA in the second three wells was digested with 5 units of the methyl-insensitive restriction enzyme MspI  to normalize the data when calculating the Global DNA Methylation Index (GDMI), and the third set of triplicates was for buffer only . All reactions were performed in a total volume of 30 μl. Prior to incubation the 96 well microtiter plate was sealed with Adhesive PCR Foil  using an ALPS 300™ , spun briefly and placed in an air incubator for 3 hours at 37°C. After incubation, the plate was spun briefly and the Adhesive PCR Foil removed. The digestion of DNA was followed by an end-fill reaction where 20 μl of biotinylation buffer containing Biotin-11-dCTP and Biotin-11-dGTP  and Sequenase  in 40 mM Tris-HCl, pH 7.5, 20 mM Tris-HCl, 50 mM NaCl) was added. The final concentration of biotinylated dCTP and dGTP and Sequenase used per well was 0.1 μM and 0.1 units respectively. The plate was sealed, spun briefly and placed into an air incubator for 30 minutes at 37°C. After the incubation period the plate was spun briefly, the Adhesive PCR Foil removed and 100 μl of Reacti-Bind™ DNA Coating Solution  was added to each well. After mixing, the plate was once again sealed and placed on an orbital platform shaker  and shaken at 150 RPM at room temperature overnight. The solution in the wells was removed and the wells were washed 4 times with TBS . The biotin was detected using HRP Neutravidin  and the DNA Detector kit . Briefly, 200 μl of the Detector Block Solution  was added to each well and the plate was incubated for 30 minutes at room temperature. After removal of the Detector Block Solution, 150 μl of Detector Block Solution containing 0.5 μg/ml (1:2000 dilution) of HRP Neutravidin was added to each well and the plate was incubated at room temperature for 30 minutes. The Detector Block/HRP Neutravidin solution was removed and the wells washed 5 times with 1xBiotin wash solution . After the final wash 150 μl of LumiGloCpGlobal the entire genome of Lambda DNA was methylated using M.SssI , a bacterial CpG methylase, which methylated all the cytosine residues that resided within a CpG dyad. Fifty micrograms of lambda genomic DNA was methylated with M.SssI for 4 hours at 37°C. The salt and enzyme were removed from the methylated lambda DNA using QIAEX II . To determine whether the lambda DNA was fully methylated 1 μg of the product was aliquoted and digested with MspI (methyl insensitive restriction enzyme) and another microgram with HpaII (a methyl sensitive restriction enzyme). The digested products were analyzed by gel electrophoresis. No fragments were observed in the HpaII digested lane. The expected size bands were measured in the MspI digested lane.To determine the analytical sensitivity of CpGlobal. The assay and the quantitation of the chemiluminescence were performed as described above. The net luminescence per DNA concentration was plotted and 25 ng of lambda DNA was determined to provide the appropriate signal to noise level for the next set of experiments.To determine the efficiency of digestion and end-fill reactions for an amount of lambda genomic DNA, we used the methyl-insensitive restriction enzyme MspI (CCGG) for which there were 328 restriction sites in the genome. The genomic DNA was aliquoted six times into a 96 well microtiter Microfluor 2 White plate. One set of triplicates were digested with MspI , the second set was treated with buffer only . A range of DNA concentrations  were assayed to determine the linearity of CpGlobal and determine the best methyl-sensitive restriction enzyme that could be applied to this assay, a series of mixtures with increasing ratios of unmethylated to methylated Lambda DNA was generated. The series of mixtures increased by 5% such that the range of methylated to unmethylated produced a broad spectrum of DNA methylation densities that varied from 100% to 0% methylated. Once the mixtures were generated, 25 ng of each DNA mix was aliquoted nine times into a 96 well microtiter Microfluor 2 White plate. To quantitate the amount of methylated Lambda DNA in each mixture the first three aliquots were digested with 5 units of a methyl-sensitive restriction enzyme  , the second three aliquots were digested with 5 units of the methyl-insensitive restriction enzyme MspI  and the third three aliquots were mixed with buffer only. The following NEB buffers  were used for different enzymes: NEBuffer1 , NEBuffer2 (BstUI and HinP1I) and NEBuffer3 (AciI). The assay and the quantitation of the chemiluminescence were performed as described above except for BstUI. This enzyme is a blunt end cutter and instead of using Sequenase during the end-fill reaction 0.1 units Klenow  was used in its stead. Here, the 3'-5'exonuclease activity of the Klenow was utilized to chew back into the blunt end-fragment and then the 5'-3'exonuclease activity would incorporate the biotinylated nucleotides.To ascertain the accuracy of CpGlobal assay for human genomic DNA was determined by measuring global DNA methylation in human male genomic DNA isolated from whole blood . A range of DNA concentrations  were assayed to determine the linearity of CpGlobal. One set of triplicates were digested with HpaII , the second set with MspI  and the third set was treated with buffer only . The assay and the quantitation of the chemiluminescence were performed as described above.The linear range of the CpGlobal with High Performance Capillary Electrophoresis (HPCE) were SW48 (CCL-231), LoVo (CCL-229), HT-29 (HTB-38) and NCI-H69 (HTB-119D). Assessment of the global DNA methylation was performed on Non-Small Cell Lung Cancer tumor cell lines from various stages of lung cancer. These included a normal lung NL20 (CRL-2503), Stage I NCI-H1703 (CRL-5889), Stage II NCI-H522 (CRL-5810), Stage IIIa NCI-H1993 (CRL-5909), Stage IIIb NCI-H1944 (CRL5907) and Stage IV metastatic liver NCI-H1755 (CRL-5892). All cells were grown in 75 cm2 cell culture flasks  in 5% CO2 at 37°C. Cells were harvested when they had reached 90% confluency. DNA was isolated using the Blood and Cell Culture DNA Midi Kit . The purity and concentration of the DNA was measured using a DU650 spectrophotometer . The quality of the DNA was determined by loading 200 ng on a 1% agarose gel to inspect for any degradation.All cell lines were purchased from ATCC  and were grown according to the manufacturer's recommendations. The cell lines that were used to compare the Twenty fresh frozen tissues samples, including lung tumors and areas of non involved lung, were obtained from a repository of biological specimens through the lung cancer Specialized Program of Research Excellence (SPORE) at the Vanderbilt Ingram Cancer Center. For each specimen, a quality control of the tissue specimen was obtained from hematoxylin and eosin stained tissue section on adjacent tissue and reviewed by a pathologist prior to analysis. De-identified clinical data elements were shared in compliance of the health insurance portability and accountability regulations. This study was approved by the local Institutional Review Board of both institutions.Normal lung DNA from young male accident victims was purchased from BioChain Institute, Inc., Hayward, CA.Approximately 0.1 gm lung tissue from biopsies of paired tumor and normal tissue from NSCLC patients was frozen in liquid nitrogen. The tissue was pulverized to a fine powder using a freezer mill 6750  according to the manufacturer's recommendations. DNA was prepared using the Blood and Cell Culture DNA midi kit . The DNA was checked for concentration and purity by reading the absorbance at 260 and 280 nM and was further tested for intactness by running 100 ng on a 1% agarose gel.Linear regression analyss was performed using EXCEL Analysis ToolPak Regression Analysis. In each case presented single predictor variable was used. R square, Adjusted R square, intercept, coefficient, standard error and F significance were generated by standard Summary Report. Residual Plots and LineFit Plots were used to check for the linearity of the regression.To determine the net luminescence, the average value for the no enzyme control was subtracted from the individual enzyme values. To calculate the global DNA methylation index (GDMI), the individual net luminescence values for the methylation sensitive enzyme were divided by the average Msp I net luminescence. All calculations were performed in Microsoft Excel.Both means and medians were calculated when comparing group differences among Normal Non-Disease, Normal Disease and Tumor samples. Only medians were used when the distributions of continuous variables were significantly skewed by small sample sizes such as different histopathological groups. Due to the potential compromise of parametric assumption, non-parametric Wilcoxon/Kruskal-Wallis tests were applied to evaluate the statistical significances for all analysis. P-value <= 0.05 was considered as statistically significant and p-value > 0.05 and <= 0.10 was regarded as border-line significant. All analyses were conducted using JMP5.1 .CpGlobal was designed to incorporate several key features: easy to use, did not require radioactivity or PCR, functioned in a 96-well microtiter plate and utilized equipment that most laboratories possessed. The design was adapted from a method that utilizes methyl-sensitive restriction enzymes [ enzymes , which wCpGlobal on Lambda DNA with the aim to determine which methyl-sensitive restriction enzyme was the best indicator in measuring global DNA methylation. Two species of lambda DNA were created: one that was unmethylated and the other that was in vitro methylated using M.SssI, a CpG methylase. Five 4 base pair methyl-sensitive restriction enzymes, AciI (CCGC), BstUI (CGCG), HpaII (CCGG), HinP1I (GCGC) and HypCH4IV (ACGT), were chosen based on their frequency within the Lambda genome and the core recognition sequence site. These enzymes were used to digest a series of Lambda DNA mixtures, which consisted of 21 points that extended from 100% to 0% methylated in 5% intervals. GDMI was generated for each 5% increment, which was compared against the theoretical methylation level. A linear regression analysis was performed for each methyl-sensitive restriction enzyme . The GDMI was calculated for human whole blood DNA and was plotted against DNA concentration. The GDMI was moderately consistent with DNA concentration , which varied in methyl content from high to low as determined by HPCE. HpaII and MspI were utilized on 100 ng of cell line genomic DNA to calculate the GDMI. The HPCE results, extracted from the publication by Paz et al [CpGlobal results presented with a good linear fit when compared to the HPCE data. In addition, we observed that the number of unmethylated HpaII sites had an inverse linear relationship with the total number of methylated cytosines (data not shown).HPCE is viewed as one of the gold standard technologies when measuring the global genomic content of 5-methylcytosine . We applaz et al  were comThe linear fit for this correlation was calculated:HPCE = -6.2 × GDMI + 7.182 = 0.77Adjusted RResidual error = 0.36.CpGlobal could be utilized as an alternative technique to one of the gold standard technologies.Considering that the HPCE data were taken from a publication to create the correlation, these results demonstrated that CpGlobal to measure changes in global DNA methylation in a cancer as it progresses from early to late stage and metastasis.We directed the assay toward measuring the association of global DNA methylation with the natural history of lung cancer. Lung cancer is measured in 5 stages, which range from 0 to IV . We measCpGlobal in lung DNA from 20 patients diagnosed with Non Small Cell Lung Cancer (NSCLC) (Table We measured the GDMI using C) Table ; an incrC) Table . The greCpGlobal could clearly distinguish a difference in the Tumor samples between Stage 1A and 1B, tumor size T1 and T2, and well differentiated and non-well differentiated tissue.To investigate further the changes in global DNA methylation in lung cancer we separated the major histopathologic characteristics assigned to the cancer patient samples into three groups  and searched for any association of the GDMI with these characteristics in the Normal Disease and Tumor samples. Nonparametric Wilcoxon/Kruskal-Wallis test was performed using the median GDMI data, which was measured in the Normal Disease and Tumor samples. No statistically significant changes were observed in global DNA methylation from the Normal Disease tissue samples for any methyl-sensitive restriction enzyme when compared against the three groups. However, the median GDMI from the Tumor tissue samples was significantly associated with all three histopathologic characteristics. In particular this was observed for HinP1I (Stage 1A versus 1B), HypCH4IV and HpaII (well versus non-well differentiated) and HinP1I and HpaII (T1 versus T2) . Here, up to 64 samples could be analyzed at once. However, the application of robotics to dispense the liquids would be more practical in this format. While the data presented in this paper were obtained from the assay where 100 ng of genomic DNA per well was used, the results showed that using as little as 3 ng could be utilized. However, we have observed that the signal to background ratio diminishes as less DNA is used (data not shown).The application of CpGlobal was able to clearly discern differences in global DNA methylation from Normal Non-Disease and Normal Disease clinical samples.The ability to access high quality tissue to perform these experiments was one of the limiting factors. While the tissue that was obtained from patients with lung cancer was highly defined and scored by experts that are knowledgeable in the histology and pathology of lung cancer, the normal tissue was obtained from car crash victims. To acquire detailed information regarding normal tissue is often difficult and limited. The variability in the tissue from accident victims can cause irreproducibility in global DNA methylation data. This may be reflected in the time and cause of death. In addition, it may explain why the median GDMI data from the Normal Non-Disease tissue were slightly higher than expected. The importance of collecting high quality tissue from normal individuals outside of blood is an on-going issue. A well-defined normal tissue bank, where sex and age matched samples could be obtained, which could go hand-in-hand with patient material, would be invaluable for diagnostic studies that seek the ability to use tissue beyond blood. An additional factor, which may have influenced the data, could be the purity of the tumor from the matched patient material. While the tumor was matched with normal it was not micro-dissected and so heterogeneity could have been an issue. Nevertheless, while the median GDMI for the tumor tissue was greater that the matched Normal Disease it was not as high as expected and this may have been attributed to heterogeneity. Given the difficulties faced in this study in acquiring truly well defined tissue, the data still remained convincing and strongly suggested CpGlobal in a disease environment provided an opportunity to ascertain the value of the technology as a research tool. In particular, how global DNA hypomethylation contributed to the instability of the genome and in the transformation of a cell from normal to disease. Genome-wide loss in methylation has been observed as one of the earliest known molecular abnormalities in human neoplasias [CpGlobal to lung cancer to delve deeper into understanding the natural history of this disease. Through the use of samples from patients diagnosed with NSCLC, two major observations from these studies were construed. The first, demonstrated that there was a statistically significant loss of global DNA methylation as the tumor progressed from Stage 1A to 1B (p = 0.08–0.006). At this early stage in the disease the only other characteristics for a pathologist to stage the lung cancer is size and grade of differentiation of the tumor. Both of these were associated with global DNA hypomethylation (p = 0.04 and p = 0.04 – 0.01 respectively). The second suggested that the histological and pathological defined normal part of the lung had incurred a loss in global DNA methylation that was statistically significant when compared with normal individuals (p = 0.039). Considering the small sample size in this study, and that by histopathologic standards the non tumor lung tissue from the cancer patients were graded as normal, global DNA hypomethylation appears to be a measurable change. Also, several studies have observed global DNA hypomethylation in other normal disease tissues such as colon, breast and ovary [The application of oplasias . This wooplasias . In thatnd ovary -24. In and ovary . It is pCpGlobal to this type of material may improve the result through combining the results of both global DNA methylation and region specific analysis using MSP. Furthermore, it may provide information as to the association of global DNA methylation and the natural history of the disease before the presence of any clinical symptoms.Exploiting these global DNA methylation changes may be of value in screening asymptomatic individuals who are at risk in developing lung cancer. An initial practical application would be to measure the global DNA methylation levels of a large sample set that contained prospective patient material. One type of sample set that could be used is from a prospective nested case control study such as the one that was utilized to measure the methylation status of the promoters from 14 genes in proximal sputum samples. Here, methyl sensitive PCR (MSP) was the technique applied to these samples. The results showed that this technique could detect changes 18 months prior to diagnosis with a 64% sensitivity and specificity . ApplicaCpGlobal is an easy-to-use technique to study the biological role of global DNA methylation in the cell. The technique can be used in almost any practical laboratory without the need for expensive equipment or highly specialized skill sets. In addition, CpGlobal can be utilized as a research tool to investigate the epigenetic phenomenon in disease, aging, diet, efficacy of drugs and in any other vertebrate or invertebrate genome where methylation is employed. The application of CpGlobal to measure the changes in global DNA methylation in lung cancer has demonstrated that there exists a distinguishing difference between Normal Non-Disease and Normal Disease tissue. Such a change should be examined further and exploited to understand the functional role of this epigenetic phenomenon with the potential aim of providing a diagnostic for asymptomatic individuals who are at risk of developing lung cancer.GDMI: global DNA methylation index; HPCE: high performance capillary electrophoresis; NSCLC: Non small cell lung cancer.The authors AA, HH, HW, SM and RGDM are co-inventors on a patent entitled "Diagnosing Diseases by Detecting DNA Methylation Changes" (United States Patent Application 20070292866). RGDM was a full time employee of AmberGen from 2005 to 2007 and is now a full time employee of Invitrogen Corporation. RGDM holds no stock or shares in AmberGen Incorporated but holds shares in Invitrogen Corporation. The article-processing charge was paid for by AmberGen Incorporated. There are no other financial or non-financial competing interests to declare that pertain to this manuscript.CpGlobal. ZL and SM isolated the DNA from the lung cancer cell lines and from the normal and tumor patient lung samples. AA performed all the experiments using CpGlobal to measure the global DNA methylation in the cell lines and patient lung samples. HH and HW performed the statistical analysis. JO was responsible for providing interpretation of the chemistry behind CpGlobal. HH and RGDM were responsible for the overall conception of CpGlobal. HH, PPM and RGDM were responsible the conception of the study and contributed to the preparation of this manuscript.AA, KIB and HG designed and performed the initial experiments with The pre-publication history for this paper can be accessed here: