Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

A phase I study of the antibody drug conjugate ASG-5ME, an SLC44A4-targeting antibody carrying auristatin E, in metastatic castration-resistant prostate cancer.

Background Antibody drug conjugates (ADC) offer the potential of maximizing efficacy while minimizing systemic toxicity. ASG-5ME, an SLC44A4-targeting antibody carrying monomethyl auristatin E (MMAE), a microtubule-disrupting agent, was investigated in men with metastatic castration resistant prostate cancer. Methods The primary objective of this phase I study was to determine maximum tolerated dose (MTD) and recommended phase II dose. Secondary objectives were safety, antitumor activity, pharmacokinetic properties, immunogenicity, and the detection of SLC44A4 on circulating tumor cells. Patients (pts) were treated among 7 dose levels every 21 days. A dose expansion phase enrolled 20 additional pts. at the MTD. Results Twenty-six and 20 pts. were treated in dose escalation and dose expansion cohorts respectively. The MTD was 2.7 mg/kg. Dose-limiting toxicities occurred in 4 pts.: grade 3 fatigue (n = 1); grade 3 abdominal pain, diarrhea and fatigue (n = 1); grade 4 neutropenia and hyponatremia and grade 3 maculopapular rash, constipation and hypoxia (n = 1); grade 3 troponin elevation without cardiac sequelae (n = 1). Fatigue and diarrhea were the most prevalent adverse events (AEs) across all cycles. Two grade 5 AEs occurred in the dose expansion cohort, each after 1 dose: 1 pt. developed grade 3 hyperglycemia, renal insufficiency and leukopenia; 1 pt. developed grade 3 hyperglycemia complicated by bacteremia. Free MMAE levels did not accumulate with repeat dosing. Of evaluable pts., 52% had either stable disease or a partial response. Conclusions Further development of ASG-5ME is not being pursued due to its narrow therapeutic index. Some toxicities were potentially related to on-target effects on normal tissue expressing the SLC44A4 protein. However, other toxicities were consistent with studies of previous MMAE-containing ADCs. Unconjugated MMAE is a less likely etiology based on prior data.

Estimating the subgroup and testing for treatment effect in a post-hoc analysis of a clinical trial with a biomarker.

We consider the problem of estimating a biomarker-based subgroup and testing for treatment effect in the overall population and in the subgroup after the trial. We define the best subgroup as the subgroup that maximizes the power for comparing the experimental treatment with the control. In the case of continuous outcome and a single biomarker, both a non-parametric method of estimating the subgroup and a method based on fitting a linear model with treatment by biomarker interaction to the data perform well. Several procedures for testing for treatment effect in all and in the subgroup are discussed. Cross-validation with two cohorts is used to estimate the biomarker cut-off to determine the best subgroup and to test for treatment effect. An approach that combines the tests in all patients and in the subgroup using Hochberg's method is recommended. This test performs well in the case when there is a subgroup with sizable treatment effect and in the case when the treatment is beneficial to everyone.

Downregulation of microRNA-21 inhibited radiation-resistance of esophageal squamous cell carcinoma.

BACKGROUND: MicroRNA-21 (miR-21) was previously reported being dysregulated in many kinds of cancer including esophageal squamous cell carcinoma (ESCC). In the present study, we aimed to investigate the role of miR-21 in ESCC, especially in its effects on radiation-sensitivity of ESCC. METHODS: Expression of miR-21 was detected in 63 pairs ESCC tumor and adjacent non-tumoral tissues using qRT-PCR, correlation between miR-21 and clinicopathological feature of ESCC was analyzed. The role of miR-21 in the proliferation, cell cycle and apoptosis of ESCC cells during irradiation were studied. RESULTS: MicroRNA-21 expression was significantly increased in ESCC tumor tissues. Expression of miR-21 was positively associated with advanced clinical stage. Under irradiation, overexpression of miR-21 increased cell proliferation and cells in S phase, and inhibited cell apoptosis of ESCC cells. In contrast, knockdown of miR-21 had an opposite effect. CONCLUSIONS: Downregulation of miR-21 inhibited the radiation-resistance of ESCC, whereas overexpression of miR-21 increased the radiation-resistance. MiR-21 is a potential novel target for developing specific treatment interventions in ESCC in future.

Urinary soluble urokinase receptor levels are elevated and pathogenic in patients with primary focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease. Recent studies have proposed that plasma soluble urokinase receptor (suPAR) might be a causative circulating factor but this proposal has caused controversy. This study aimed to measure urinary suPAR levels in patients with primary FSGS and its significance in the pathogenesis of FSGS. METHODS: Sixty-two patients with primary FSGS, diagnosed between January 2006 and January 2012, with complete clinical and pathologic data were enrolled, together with disease and normal controls. Urinary suPAR levels were measured using commercial ELISA kits and were corrected by urinary creatinine (Cr). The associations between urinary suPAR levels and clinical data at presentation and during follow up were analyzed. Conditionally immortalized human podocytes were used to study the effect of urinary suPAR on activating ß3 integrin detected by AP5 staining. RESULTS: The urinary suPAR level of patients with primary FSGS (500.56, IQR 262.78 to 1,059.44 pg/µmol Cr) was significantly higher than that of patients with minimal change disease (307.86, IQR 216.54 to 480.18 pg/µmol Cr, P = 0.033), membranous nephropathy (250.23, IQR 170.37 to 357.59 pg/µmol Cr, P <0.001), secondary FSGS (220.45, IQR 149.38 to 335.54 pg/µmol Cr, P <0.001) and normal subjects (183.59, IQR 103.92 to 228.78 pg/µmol Cr, P <0.001). The urinary suPAR level of patients with cellular variant was significantly higher than that of patients with tip variant. The urinary suPAR level in the patients with primary FSGS was positively correlated with 24-hour urine protein (r = 0.287, P = 0.024). During follow up, the urinary suPAR level of patients with complete remission decreased significantly (661.19, IQR 224.32 to 1,115.29 pg/µmol Cr versus 217.68, IQR 121.77 to 415.55 pg/µmol Cr, P = 0.017). The AP5 signal was strongly induced along the cell membrane when human differentiated podocytes were incubated with the urine of patients with FSGS at presentation, and the signal could be reduced by a blocking antibody specific to uPAR. CONCLUSIONS: Urinary suPAR was specifically elevated in patients with primary FSGS and was associated with disease severity. The elevated urinary suPAR could activate ß3 integrin on human podocytes.

Plasma soluble urokinase receptor levels are increased but do not distinguish primary from secondary focal segmental glomerulosclerosis.

In this study, we measured soluble urokinase receptor levels, a possible permeability factor, in the plasma of patients with primary focal segmental glomerulosclerosis (FSGS) and determined their association with clinical and pathological data in 74 patients with primary FSGS. Healthy donors and patients with minimal change disease, membranous nephropathy, and secondary FSGS were used as controls. The plasma-soluble urokinase receptor levels, measured by commercial ELISA kits, of patients with primary FSGS (median: 2923, interquartile range 2205-4360 pg/ml) were significantly higher than those of patients with minimal change disease (median 2050 pg/ml), membranous nephropathy (median 2029 pg/ml), and normal individuals (median 1739 pg/ml). There was no significant difference in plasma-soluble urokinase receptor levels between the 74 patients with primary and 14 patients with secondary FSGS. The soluble urokinase receptor levels increased in the order of tip variant, to a not otherwise specified variant and a cellular variant. The soluble urokinase receptor levels were significantly but negatively correlated with creatinine clearance at presentation but positively correlated with crescent formation in patients with primary FSGS. During follow-up, receptor levels decreased significantly in patients with complete remission. Thus, plasma-soluble urokinase receptor levels did not differentiate primary and secondary FSGS, and although significantly elevated in FSGS, they showed considerable overlap with other glomerular diseases.

Early mobilization in critically ill children: a survey of Canadian practice.

OBJECTIVES: While early mobilization is safe and enhances functional recovery in critically ill adults, rehabilitation practices in critically ill children are not well characterized. The objective of this study was to evaluate the knowledge, perceptions, and stated practices of early mobilization among physicians and physiotherapists practicing in Canadian pediatric critical care units. DESIGN AND MEASUREMENTS: A self-administered survey was mailed to 102 physicians and 35 physiotherapists. Survey domains included barriers to early mobilization, the timing, nature and thresholds for rehabilitation, and staffing workload. We assessed for associations using chi-square tests. MAIN RESULTS: The overall response rate was 64.2% (88 of 137), representing 59.8% (61 of 102) physicians and 77.1% (27 of 35) physiotherapists, respectively. Key institutional barriers to early mobilization included a lack of practice guidelines (75.4% physician, 48.1% physiotherapist respondents; p = 0.01) and the need for physician orders prior to initiating physiotherapy (26.2% physician vs 55.6% physiotherapist, p = 0.008). Only 3.4% of respondents reported having local guidelines for early mobilization. Conflicting perceptions regarding the clinical thresholds for early mobilization and the safety of early mobilization were the most commonly reported patient-level barriers. Increasing illness severity was associated with decreased clinician comfort with early mobilization. Respiratory physiotherapy and passive range of motion were the most frequently applied rehabilitation interventions (77.8%), while pregait physiotherapy and ambulation were only sometimes or infrequently (70.4%) used. The type and extent of physiotherapy varied depending on the time of day and week. CONCLUSIONS: There are numerous perceived institutional, patient- and provider-level barriers to early mobilization in Canadian pediatric critical care units, and diverse opinions on the appropriateness of early mobilization. Limited awareness of existing literature and the lack of practice guidelines on early mobilization are not surprising in light of the paucity of pediatric-specific evidence. These results strongly support the need for further research, evaluating the feasibility, safety, and efficacy of early mobilization in critically ill children.

Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use.

The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.

Postoperative B-type natriuretic peptide for prediction of major cardiac events in patients undergoing noncardiac surgery: systematic review and individual patient meta-analysis.

BACKGROUND: It is unclear whether postoperative B-type natriuretic peptides (i.e., BNP and N-terminal proBNP) can predict cardiovascular complications in noncardiac surgery. METHODS: The authors undertook a systematic review and individual patient data meta-analysis to determine whether postoperative BNPs predict postoperative cardiovascular complications at 30 and 180 days or more. RESULTS: The authors identified 18 eligible studies (n = 2,051). For the primary outcome of 30-day mortality or nonfatal myocardial infarction, BNP of 245 pg/ml had an area under the curve of 0.71 (95% CI, 0.64-0.78), and N-terminal proBNP of 718 pg/ml had an area under the curve of 0.80 (95% CI, 0.77-0.84). These thresholds independently predicted 30-day mortality or nonfatal myocardial infarction (adjusted odds ratio [AOR] 4.5; 95% CI, 2.74-7.4; P < 0.001), mortality (AOR, 4.2; 95% CI, 2.29-7.69; P < 0.001), cardiac mortality (AOR, 9.4; 95% CI, 0.32-254.34; P < 0.001), and cardiac failure (AOR, 18.5; 95% CI, 4.55-75.29; P < 0.001). For greater than or equal to 180-day outcomes, natriuretic peptides independently predicted mortality or nonfatal myocardial infarction (AOR, 3.3; 95% CI, 2.58-4.3; P < 0.001), mortality (AOR, 2.2; 95% CI, 1.67-86; P < 0.001), cardiac mortality (AOR, 2.1; 95% CI, 0.05-1,385.17; P < 0.001), and cardiac failure (AOR, 3.5; 95% CI, 1.0-9.34; P = 0.022). Patients with BNP values of 0-250, greater than 250-400, and greater than 400 pg/ml suffered the primary outcome at a rate of 6.6, 15.7, and 29.5%, respectively. Patients with N-terminal proBNP values of 0-300, greater than 300-900, and greater than 900 pg/ml suffered the primary outcome at a rate of 1.8, 8.7, and 27%, respectively. CONCLUSIONS: Increased postoperative BNPs are independently associated with adverse cardiac events after noncardiac surgery.

Impact of tuberculosis on mortality among HIV-infected patients receiving antiretroviral therapy in Uganda: a prospective cohort analysis.

BACKGROUND: Tuberculosis (TB) disease affects survival among HIV co-infected patients on antiretroviral therapy (ART). Yet, the magnitude of TB disease on mortality is poorly understood. METHODS: Using a prospective cohort of 22,477 adult patients who initiated ART between August 2000 and June 2009 in Uganda, we assessed the effect of active pulmonary TB disease at the initiation of ART on all-cause mortality using a Cox proportional hazards model. Propensity score (PS) matching was used to control for potential confounding. Stratification and covariate adjustment for PS and not PS-based multivariable Cox models were also performed. RESULTS: A total of 1,609 (7.52%) patients had active pulmonary TB at the start of ART. TB patients had higher proportions of being male, suffering from AIDS-defining illnesses, having World Health Organization (WHO) disease stage III or IV, and having lower CD4 cell counts at baseline (p < 0.001). The percentages of death during follow-up were 10.47% and 6.38% for patients with and without TB, respectively. The hazard ratio (HR) for mortality comparing TB to non-TB patients using 1,686 PS-matched pairs was 1.37 (95% confidence interval [CI]: 1.08 - 1.75), less marked than the crude estimate (HR = 1.74, 95% CI: 1.49 - 2.04). The other PS-based methods and not PS-based multivariable Cox model produced similar results. CONCLUSIONS: After controlling for important confounding variables, HIV patients who had TB at the initiation of ART in Uganda had an approximate 37% increased hazard of overall mortality relative to non-TB patients.

The aging Canadian population and hospitalizations for acute myocardial infarction: projection to 2020.

BACKGROUND: The risk of experiencing an acute myocardial infarction (AMI) increases with age and Canada's population is aging. The objective of this analysis was to examine trends in the AMI hospitalization rate in Canada between 2002 and 2009 and to estimate the potential increase in the number of AMI hospitalizations over the next decade. METHODS: Aggregated data on annual AMI hospitalizations were obtained from the Canadian Institute for Health Information for all provinces and territories, except Quebec, for 2002/03 and 2009/10. Using these data in a Poisson regression model to control for age, gender and year, the rate of AMI hospitalizations was extrapolated between 2010 and 2020. The extrapolated rate and Statistics Canada population projections were used to estimate the number of AMI hospitalizations in 2020. RESULTS: The rates of AMI hospitalizations by gender and age group showed a decrease between 2002 and 2009 in patients aged ≥ 65 years and relatively stable rates in those aged < 64 years in both males and females. However, the total number of AMI hospitalizations in Canada (excluding Quebec) is projected to increase by 4667 from 51847 in 2009 to 56514 in 2020, a 9.0% increase. Inflating this number to account for the unavailable Quebec data results in an increase of approximately 6200 for the whole of Canada. This would amount to an additional cost of between $46 and $54 million and sensitivity analyses indicate that it could be between $36 and $65 million. CONCLUSIONS: Despite projected decreasing or stable rates of AMI hospitalization, the number of hospitalizations is expected to increase substantially as a result of the aging of the Canadian population. The cost of these hospitalizations will be substantial. An increase of this extent in the number of AMI hospitalizations and the ensuing costs would significantly impact the already over-stretched Canadian healthcare system.

How well are journal and clinical article characteristics associated with the journal impact factor? a retrospective cohort study.

OBJECTIVE: Journal impact factor (JIF) is often used as a measure of journal quality. A retrospective cohort study determined the ability of clinical article and journal characteristics, including appraisal measures collected at the time of publication, to predict subsequent JIFs. METHODS: Clinical research articles that passed methods quality criteria were included. Each article was rated for relevance and newsworthiness by 3 to 24 physicians from a panel of more than 4,000 practicing clinicians. The 1,267 articles (from 103 journals) were divided 60∶40 into derivation (760 articles) and validation sets (507 articles), representing 99 and 88 journals, respectively. A multiple regression model was produced determining the association of 10 journal and article measures with the 2007 JIF. RESULTS: Four of the 10 measures were significant in the regression model: number of authors, number of databases indexing the journal, proportion of articles passing methods criteria, and mean clinical newsworthiness scores. With the number of disciplines rating the article, the 5 variables accounted for 61% of the variation in JIF (R(2) = 0.607, 95% CI 0.444 to 0.706, P<0.001). CONCLUSION: For the clinical literature, measures of scientific quality and clinical newsworthiness available at the time of publication can predict JIFs with 60% accuracy.

Development of a new chemo-enzymatic catalytic route for synthesis of (S)- 2-chlorophenylglycine.

(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant EsLeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (BmGDH) for NADH regeneration, EsLeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C.

Comparing methods to estimate treatment effects on a continuous outcome in multicentre randomized controlled trials: a simulation study.

BACKGROUND: Multicentre randomized controlled trials (RCTs) routinely use randomization and analysis stratified by centre to control for differences between centres and to improve precision. No consensus has been reached on how to best analyze correlated continuous outcomes in such settings. Our objective was to investigate the properties of commonly used statistical models at various levels of clustering in the context of multicentre RCTs. METHODS: Assuming no treatment by centre interaction, we compared six methods (ignoring centre effects, including centres as fixed effects, including centres as random effects, generalized estimating equation (GEE), and fixed- and random-effects centre-level analysis) to analyze continuous outcomes in multicentre RCTs using simulations over a wide spectrum of intraclass correlation (ICC) values, and varying numbers of centres and centre size. The performance of models was evaluated in terms of bias, precision, mean squared error of the point estimator of treatment effect, empirical coverage of the 95% confidence interval, and statistical power of the procedure. RESULTS: While all methods yielded unbiased estimates of treatment effect, ignoring centres led to inflation of standard error and loss of statistical power when within centre correlation was present. Mixed-effects model was most efficient and attained nominal coverage of 95% and 90% power in almost all scenarios. Fixed-effects model was less precise when the number of centres was large and treatment allocation was subject to chance imbalance within centre. GEE approach underestimated standard error of the treatment effect when the number of centres was small. The two centre-level models led to more variable point estimates and relatively low interval coverage or statistical power depending on whether or not heterogeneity of treatment contrasts was considered in the analysis. CONCLUSIONS: All six models produced unbiased estimates of treatment effect in the context of multicentre trials. Adjusting for centre as a random intercept led to the most efficient treatment effect estimation across all simulations under the normality assumption, when there was no treatment by centre interaction.

The efficacy of botulinum toxin type A in managing chronic musculoskeletal pain: a systematic review and meta analysis.

BACKGROUND: Botulinum toxin type A (BoNTA) is a neurotoxin that acts by inhibiting the release of neurotransmitters acetylcholine at neuromuscular junctions, thus reducing muscular contractions. Recent evidence suggests that BoNTA can reduce nociceptive activities of sensory neurons in animal models by inhibiting release of certain neuropeptides. Despite the therapeutic benefit of BoNTA in alleviating painful muscle spasms, its efficacy in other musculoskeletal pain conditions is less clear. OBJECTIVE: We aim to examine the efficacy of BoNTA in reducing chronic musculoskeletal pain. METHODS: Studies for inclusion in our report were identified using MEDLINE, EMBASE, PUBMED, Cochrane Central Register of Controlled Trials, CINAHL, and reference lists of relevant articles. Studies were considered eligible for inclusion if they were randomized controlled trials (RCTs), evaluating the efficacy of BoNTA injections in pain reduction. All studies were assessed and data were abstracted independently by paired reviewers. The outcome measures were baseline and final pain scores as assessed by the patients. The internal validity of trials was assessed with the Jadad scale. Disagreements were resolved through discussions. MAIN RESULTS: Twenty-one studies were included in the systematic review and 15 of them were included in the final meta-analysis. There was a total of 706 patients in the meta-analysis, represented from trials of plantar fasciitis (n = 1), tennis elbow (n = 2), shoulder pain (n = 1), whiplash (n = 3), and myofascial pain (n = 8). Overall, there was a small to moderate pain reduction among BoNTA patients when compared to control (SMD = -0.27, 95% CI: -0.44 to -0.11). When the results were analyzed in subgroups, only tennis elbow (SMD = -0.44, 95% CI: -0.86 to -0.01) and plantar fasciitis (SMD = -1.04, 95% CI: -1.68 to -0.40) demonstrated significant pain relief. Although not in the meta-analysis, one back pain study also demonstrated positive results for BoNTA. Lastly, BoNTA was effective when used at ≥ 25 units per anatomical site or after a period ≥ 5 weeks. CONCLUSION: In our meta-analysis, BoNTA had a small to moderate analgesic effect in chronic musculoskeletal pain conditions. It was particularly effective in plantar fasciitis, tennis elbow, and back pain, but not in whiplash or shoulder pain patients. However, more evidence is required before definitive conclusions can be drawn. On the other hand, there is convincing evidence that BoNTA lacks strong analgesic effects in patients with myofascial pain syndrome. A general dose-dependent and temporal response with BoNTA injections was also observed.

Bayesian adjustment for exposure misclassification in case-control studies.

Poor measurement of explanatory variables occurs frequently in observational studies. Error-prone observations may lead to biased estimation and loss of power in detecting the impact of explanatory variables on the response. We consider misclassified binary exposure in the context of case-control studies, assuming the availability of validation data to inform the magnitude of the misclassification. A Bayesian adjustment to correct the misclassification is investigated. Simulation studies show that the Bayesian method can have advantages over non-Bayesian counterparts, particularly in the face of a rare exposure, small validation sample sizes, and uncertainty about whether exposure misclassification is differential or non-differential. The method is illustrated via application to several real studies.

A tutorial on pilot studies: the what, why and how.

Pilot studies for phase III trials - which are comparative randomized trials designed to provide preliminary evidence on the clinical efficacy of a drug or intervention - are routinely performed in many clinical areas. Also commonly know as "feasibility" or "vanguard" studies, they are designed to assess the safety of treatment or interventions; to assess recruitment potential; to assess the feasibility of international collaboration or coordination for multicentre trials; to increase clinical experience with the study medication or intervention for the phase III trials. They are the best way to assess feasibility of a large, expensive full-scale study, and in fact are an almost essential pre-requisite. Conducting a pilot prior to the main study can enhance the likelihood of success of the main study and potentially help to avoid doomed main studies. The objective of this paper is to provide a detailed examination of the key aspects of pilot studies for phase III trials including: 1) the general reasons for conducting a pilot study; 2) the relationships between pilot studies, proof-of-concept studies, and adaptive designs; 3) the challenges of and misconceptions about pilot studies; 4) the criteria for evaluating the success of a pilot study; 5) frequently asked questions about pilot studies; 7) some ethical aspects related to pilot studies; and 8) some suggestions on how to report the results of pilot investigations using the CONSORT format.

Water and sewage systems, socio-demographics, and duration of residence associated with endemic intestinal infectious diseases: a cohort study.

BACKGROUND: Studies of water-related gastrointestinal infections are usually directed at outbreaks. Few have examined endemic illness or compared rates across different water supply and sewage disposal systems. We conducted a cohort study of physician visits and hospitalizations for endemic intestinal infectious diseases in a mixed rural and urban community near Vancouver, Canada, with varied and well-characterized water and sewage systems. METHODS: Cohort members and their disease events were defined via universal health insurance data from 1995 through 2003. Environmental data were derived from municipal, provincial, and federal government sources. Logistic regression was used to examine associations between disease events and water and sewage systems, socio-demographic characteristics, and temporal factors. RESULTS: The cohort included 126,499 individuals and approximately 190,000,000 person-days. Crude incidence rates were 1,353 physician visits and 33.8 hospitalizations for intestinal infectious diseases per 100,000 person-years. Water supply chlorination was associated with reduced physician visit incidence (OR: 0.92, 95% CI 0.85-1.0). Two water systems with the highest proportions of surface water had increased incidence (ORs: 1.57, 95% CI 1.39-1.78; and 1.45, 95% CI 1.28-1.64). Private well water and well depth were not associated with increased risk, likely because of residents' awareness of and attention to water quality. There was increased crude incidence with increasing precipitation in the population served by surface water supplies, but this trend did not remain with adjustment for other variables. Municipal sewer systems were associated with increased risk (OR: 1.26, 95% CI 1.14-1.38). Most socio-demographic variables had predicted associations with risk: higher rates in females, in the very young and the elderly, and in residents of low income areas. Increased duration of area residence was associated with reduced risk (OR, duration ≥ 6 years: 0.69, 95% CI 0.60-0.80 vs. < 1 year: 1.16, 95% CI 1.03-1.30). CONCLUSIONS: This large cohort study, with objective data on exposures and outcomes, demonstrated associations between endemic infectious intestinal diseases and factors related to water supply, sewage disposal, socio-demographics, and duration of residency. The results did not always follow prior expectations based on studies examining outbreaks and single systems, and underscore the importance of studying factors associated with endemic disease across water and sewage system types.

Public opinions about participating in health research.

OBJECTIVES: Privacy legislation has limited options for recruiting subjects to health studies. Policy changes are motivated by assumptions about public attitudes towards participation, yet surveys of attitudes have rarely been done. We investigated public willingness to participate in health research and how willingness was affected by various factors. METHODS: A survey of adults randomly selected from the telephone directory was conducted in British Columbia, Canada. Mailed self-administered questionnaires asked about willingness to participate in health research and the influence on willingness of the method of subject selection, the organization making the contact, and other factors. RESULTS: There were 1,477 respondents (58% of eligible); 85% were willing to participate in health research at least sometimes. The organization making the contact influenced comfort about participation: 10% of respondents felt uncomfortable if contacted by a university, 12% if by a hospital, 26% if by government, and 55% if by private research firms. Factors most positively influencing choice to participate were future health benefits to society (87%) and oneself (87%), and receiving a copy of the study results (81%). CONCLUSIONS: Participation in health research appears to be viewed favourably by members of the public, and participation may be highest when university or hospital-based researchers are able to contact subjects directly using information from government databases.