Bleeding Risk Prediction in Patients Treated with Antithrombotic Drugs According to the Anatomic Site of Bleeding, Indication for Treatment, and Time Since Treatment Initiation.

Background Reasons for the relatively poor performance of bleeding prediction models are not well understood but may relate to differences in predictors for various anatomical sites of bleeding. Methods We pooled individual participant data from four randomized controlled trials of antithrombotic therapy in patients with coronary and peripheral artery diseases, embolic stroke of undetermined source (ESUS), or atrial fibrillation. We examined discrimination and calibration of models for any major bleeding, major gastrointestinal (GI) bleeding, and intracranial hemorrhage (ICH), according to the time since initiation of antithrombotic therapy, and indication for antithrombotic therapy. Results Of 57,813 patients included, 1,948 (3.37%) experienced major bleeding, including 717 (1.24%) major GI bleeding and 274 (0.47%) ICH. The model derived to predict major bleeding at 1 year from any site (c-index, 0.69, 95% confidence interval [CI], 0.68-0.71) performed similarly when applied to predict major GI bleeding (0.71, 0.69-0.74), but less well to predict ICH (0.64, 0.61-0.69). Models derived to predict GI bleeding (0.75, 0.74-0.78) and ICH (0.72, 0.70-0.79) performed better than the general major bleeding model. Discrimination declined over time since the initiation of antithrombotic treatment, stabilizing at approximately 2 years for any major bleeding and major GI bleeding and 1 year for ICH. Discrimination was best for the model predicting ICH in the ESUS population (0.82, 0.78-0.92) and worst for the model predicting any major bleeding in the coronary and peripheral artery disease population (0.66, 0.65-0.69). Conclusion Performance of risk prediction models for major bleeding is affected by site of bleeding, time since initiation of antithrombotic therapy, and indication for antithrombotic therapy.

An evolutionary algorithm for the direct optimization of covariate balance between nonrandomized populations.

Matching reduces confounding bias in comparing the outcomes of nonrandomized patient populations by removing systematic differences between them. Under very basic assumptions, propensity score (PS) matching can be shown to eliminate bias entirely in estimating the average treatment effect on the treated. In practice, misspecification of the PS model leads to deviations from theory and matching quality is ultimately judged by the observed post-matching balance in baseline covariates. Since covariate balance is the ultimate arbiter of successful matching, we argue for an approach to matching in which the success criterion is explicitly specified and describe an evolutionary algorithm to directly optimize an arbitrary metric of covariate balance. We demonstrate the performance of the proposed method using a simulated dataset of 275,000 patients and 10 matching covariates. We further apply the method to match 250 patients from a recently completed clinical trial to a pool of more than 160,000 patients identified from electronic health records on 101 covariates. In all cases, we find that the proposed method outperforms PS matching as measured by the specified balance criterion. We additionally find that the evolutionary approach can perform comparably to another popular direct optimization technique based on linear integer programming, while having the additional advantage of supporting arbitrary balance metrics. We demonstrate how the chosen balance metric impacts the statistical properties of the resulting matched populations, emphasizing the potential impact of using nonlinear balance functions in constructing an external control arm. We release our implementation of the considered algorithms in Python.

Clinical characteristics and disease outcomes in non-diabetic chronic kidney disease: retrospective analysis of a US healthcare claims database.

BACKGROUND: The observational, real-world evidence FLIEDER study aimed to describe patient clinical characteristics and investigate clinical outcomes in non-diabetic patients with chronic kidney disease (CKD) using data collected from routine clinical practice in the United States. METHODS: Between 1 January, 2008-31 December, 2018, individuals aged ≥ 18 years, with non-diabetic, stage 3-4 CKD were indexed in the Optum® Clinformatics® Data Mart US healthcare claims database using International Classification of Diseases-9/10 codes for CKD or by laboratory values (estimated glomerular filtration rate [eGFR] 15-59 mL/min/1.73 m2). The primary outcomes were hospitalization for heart failure, a composite kidney outcome of end-stage kidney disease/kidney failure/need for dialysis and worsening of CKD stage from baseline. The effects of the intercurrent events of a sustained post-baseline decline in eGFR ≥ 30%, ≥ 40%, and ≥ 57% on the subsequent risk of the primary outcomes were also assessed. RESULTS: In the main study cohort (N = 504,924), median age was 75.0 years, and 60.5% were female. Most patients (94.7%) had stage 3 CKD at index. Incidence rates for hospitalization for heart failure, the composite kidney outcome, and worsening of CKD stage from baseline were 4.0, 10.3, and 4.4 events/100 patient-years, respectively. The intercurrent event analysis demonstrated that a relative decline in kidney function from baseline significantly increased the risk of cardiorenal events. CONCLUSIONS: This real-world study highlights that patients with non-diabetic CKD are at high risk of serious adverse clinical outcomes, and that this risk is amplified in patients who experienced greater post-baseline eGFR decline.

Identification of International Society on Thrombosis and Haemostasis major and clinically relevant non-major bleed events from electronic health records: a novel algorithm to enhance data utilisation from real-world sources.

Introduction: In randomised controlled trials (RCTs), bleeding outcomes are often assessed using definitions provided by the International Society on Thrombosis and Haemostasis (ISTH). Information relating to bleeding events in real-world evidence (RWE) sources are not identified using these definitions. To assist with accurate comparisons between clinical trials and real-world studies, algorithms are required for the identification of ISTH-defined bleeding events in RWE sources. Objectives: To present a novel algorithm to identify ISTH-defined major and clinically-relevant non-major (CRNM) bleeding events in a US Electronic Health Record (EHR) database. Methods: The ISTH definition for major bleeding was divided into three subclauses: fatal bleeds, critical organ bleeds and symptomatic bleeds associated with haemoglobin reductions. Data elements from EHRs required to identify patients fulfilling these subclauses (algorithm components) were defined according to International Classification of Diseases, 9th and 10th Revisions, Clinical Modification disease codes that describe key bleeding events. Other data providing context to bleeding severity included in the algorithm were: 'interaction type' (diagnosis in the inpatient or outpatient setting), 'position' (primary/discharge or secondary diagnosis), haemoglobin values from laboratory tests, blood transfusion codes and mortality data. Results: In the final algorithm, the components were combined to align with the subclauses of ISTH definitions for major and CRNM bleeds. A matrix was proposed to guide identification of ISTH bleeding events in the EHR database. The matrix categorises bleeding events by combining data from algorithm components, including: diagnosis codes, 'interaction type', 'position', decreases in haemoglobin concentrations (≥ 2 g/dL over 48 hours) and mortality. Conclusions: The novel algorithm proposed here identifies ISTH major and CRNM bleeding events that are commonly investigated in RCTs in a real-world EHR data source. This algorithm could facilitate comparison between the frequency of bleeding outcomes recorded in clinical trials and RWE. Validation of algorithm performance is in progress.

Worsening of kidney function in patients with atrial fibrillation and chronic kidney disease: evidence from the real-world CALLIPER study.

OBJECTIVE: Evidence is needed on the impact of anticoagulation therapy on kidney function in patients with atrial fibrillation (AF). The objective of this analysis, which is part of the CALLIPER study, was to investigate the risk of worsening kidney function with rivaroxaban 15 mg once daily compared with warfarin in patients with AF and moderate-to-severe chronic kidney disease (CKD) in routine clinical practice in the United States. METHODS: CALLIPER was an observational, retrospective, new-user cohort study. Adult patients with AF in the US IBM Watson MarketScan databases who newly initiated anticoagulation with rivaroxaban 15 mg once daily or warfarin between January 2013 and December 2017 were included. Comparative analysis was performed using Cox proportional hazards regression after adjustment for potential confounding by the stabilized inverse probability of treatment weighting approach and propensity score matching. One of the main study outcomes was worsening kidney function (composite of progression to CKD stage 5, kidney failure, or need for dialysis), besides traditional AF-related outcomes. RESULTS: The cohort included 7368 patients: 5903 (80.1%) initiating warfarin and 1465 (19.9%) initiating rivaroxaban 15 mg once daily. Rivaroxaban 15 mg was associated with a significant 47% reduction in the risk of worsening kidney function versus warfarin (hazard ratio 0.53; 95% confidence interval 0.35-0.78). Similar results were observed in the subgroup of patients with type 2 diabetes. CONCLUSIONS: Rivaroxaban 15 mg may be associated with a lower risk of worsening kidney function as compared with warfarin in the atrial fibrillation population with moderate-to-severe CKD. TRIAL REGISTRATION NUMBER: NCT03359876.

Serological response to nifurtimox in adult patients with chronic Chagas disease: An observational comparative study in Argentina.

Nifurtimox is indicated in Chagas disease but determining its effectiveness in chronic disease is hindered by the length of time needed to demonstrate negative serological conversion. We manually reviewed long-term follow-up data from hospital records of patients with chronic Chagas disease (N = 1,497) in Argentina diagnosed during 1967-1980. All patients were aged ≥18 years at diagnosis and were either treated with nifurtimox (n = 968) or received no antitrypanosomal treatment (n = 529). The primary endpoint was negative seroconversion (the "event"), defined as a change from positive to negative in the serological or parasitological laboratory test used at diagnosis. Time to event was from baseline visit to date of endpoint event or censoring. The effectiveness of nifurtimox versus no treatment was estimated with Cox proportional hazard regression using propensity scores with overlap weights to calculate the hazard ratio and 95% confidence interval. The nifurtimox group was younger than the untreated group (mean, 32.4 vs. 40.3 years), with proportionally fewer females (47.9% vs. 60.1%), and proportionally more of the nifurtimox group than the untreated group had clinical signs and symptoms of Chagas disease at diagnosis (28.9% vs. 14.0%). Median maximum daily dose of nifurtimox was 8.0 mg/kg/day (interquartile range [IQR]: 8.0-9.0) and median treatment duration was 44 days (IQR: 1-90). Median time to event was 2.1 years (IQR: 1.0-4.5) for nifurtimox-treated and 2.4 years (IQR: 1.0-4.2) for untreated patients. Accounting for potential confounders, the estimated hazard ratio (95% confidence interval) for negative seroconversion was 2.22 (1.61-3.07) favoring nifurtimox. Variable treatment regimens and follow-up duration, and an uncommonly high rate of spontaneous negative seroconversion, complicate interpretation of this epidemiological study, but with the longest follow-up and largest cohort analyzed to date it lends weight to the benefit of nifurtimox in adults with chronic Chagas disease. Trial registration: The study protocol was registered at ClinicalTrials.gov: NCT03784391.

Digital Tracking of Physical Activity, Heart Rate, and Inhalation Behavior in Patients With Pulmonary Arterial Hypertension Treated With Inhaled Iloprost: Observational Study (VENTASTEP).

BACKGROUND: Pulmonary arterial hypertension restricts the ability of patients to perform routine physical activities. As part of pulmonary arterial hypertension treatment, inhaled iloprost can be administered via a nebulizer that tracks inhalation behavior. Pulmonary arterial hypertension treatment is guided by intermittent clinical measurements, such as 6-minute walk distance, assessed during regular physician visits. Continuous digital monitoring of physical activity may facilitate more complete assessment of the impact of pulmonary arterial hypertension on daily life. Physical activity tracking with a wearable has not yet been assessed with simultaneous tracking of pulmonary arterial hypertension medication intake. OBJECTIVE: We aimed to digitally track the physical parameters of patients with pulmonary arterial hypertension who were starting treatment with iloprost using a Breelib nebulizer. The primary objective was to investigate correlations between changes in digital physical activity measures and changes in traditional clinical measures and health-related quality of life over 3 months. Secondary objectives were to evaluate inhalation behavior, adverse events, and changes in heart rate and sleep quality. METHODS: We conducted a prospective, multicenter observational study of adults with pulmonary arterial hypertension in World Health Organization functional class III who were adding inhaled iloprost to existing pulmonary arterial hypertension therapy. Daily distance walked, step count, number of standing-up events, heart rate, and 6-minute walk distance were digitally captured using smartwatch (Apple Watch Series 2) and smartphone (iPhone 6S) apps during a 3-month observation period (which began when iloprost treatment began). Before and at the end of the observation period (within 2 weeks), we also evaluated 6-minute walk distance, Borg dyspnea, functional class, B-type natriuretic peptide (or N-terminal pro-B-type natriuretic peptide) levels, health-related quality of life (EQ-5D questionnaire), and sleep quality (Pittsburgh Sleep Quality Index). RESULTS: Of 31 patients, 18 were included in the full analysis (observation period: median 91.5 days, IQR 88.0 to 92.0). Changes from baseline in traditional and digital 6-minute walk distance were moderately correlated (r=0.57). Physical activity (daily distance walked: median 0.4 km, IQR -0.2 to 1.9; daily step count: median 591, IQR -509 to 2413) and clinical measures (traditional 6-minute walk distance: median 26 m, IQR 0 to 40) changed concordantly from baseline to the end of the observation period. Health-related quality of life showed little change. Total sleep score and resting heart rate slightly decreased. Distance walked and step count showed short-term increases after each iloprost inhalation. No new safety signals were identified (safety analysis set: n=30). CONCLUSIONS: Our results suggest that despite challenges, parallel monitoring of physical activity, heart rate, and iloprost inhalation is feasible in patients with pulmonary arterial hypertension and may complement traditional measures in guiding treatment; however, the sample size of this study limits generalizability. TRIAL REGISTRATION: ClinicalTrials.gov NCT03293407; https://clinicaltrials.gov/ct2/show/NCT03293407. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/12144.

Distinguishing atrial fibrillation from sinus rhythm using commercial pulse detection systems: The non-interventional BAYathlon study.

OBJECTIVE: Early diagnosis of atrial fibrillation (AFib) is a priority for stroke prevention. We sought to test four commercial pulse detection systems (CPDSs) for ability to distinguish AFib from normal sinus rhythm using a published algorithm (Zhou et al., PLoS One 2015;10:e0136544), compared with visual diagnosis by electrocardiogram inspection. METHODS: BAYathlon was a prospective, non-interventional, single-centre study. Adult cardiology patients with documented AFib or sinus rhythm who were due to have a routine 5-min electrocardiogram were randomized to undergo a parallel 5-min pulse assessment with a Polar V800, eMotion Faros 360, TomTom heart rate monitor, or Adidas miCoach Smart Run. RESULTS: 144 patients (73 with AFib, 71 with sinus rhythm (based on electrocardiograms); median age: 73 years; 53.5% male) were analysed. Algorithm sensitivities (primary endpoint) and specificities for AFib when applied to CPDS recordings were 93.3% and 94.1% with the Polar V800, 90.0% and 84.2% with the eMotion Faros 360, and 0% and 100% with the other CPDSs (analysis period: 127 heart rate signals + 2 min). When applied to routine electrocardiograms, the algorithm correctly detected AFib in 71/73 patients. Different analysis periods (127 heart rate signals +1 or 3 min) only slightly changed the sensitivities with the Polar V800 and eMotion Faros 360 and had no effect on the sensitivities with the other CPDSs. CONCLUSION: AFib screening using the applied algorithm is feasible with the Polar V800 and eMotion Faros 360 (which provide RR interval data) but not with the other CPDSs (which provide pre-processed heart rate time series).ClinicalTrials.gov identifier: NCT02875106.

Switching to riociguat versus maintenance therapy with phosphodiesterase-5 inhibitors in patients with pulmonary arterial hypertension (REPLACE): a multicentre, open-label, randomised controlled trial.

BACKGROUND: Riociguat and phosphodiesterase-5 inhibitors (PDE5i), approved for the treatment of pulmonary arterial hypertension (PAH), act on the same pathway via different mechanisms. Riociguat might be an alternative option for patients with PAH who do not respond sufficiently to treatment with PDE5i, but comparisons of the potential benefits of riociguat and PDE5i in these patients are needed. The aim of this trial was to assess the effects of switching to riociguat from PDE5i therapy versus continued PDE5i therapy in patients with PAH at intermediate risk of 1-year mortality. METHODS: Riociguat rEplacing PDE5i therapy evaLuated Against Continued PDE5i thErapy (REPLACE) was an open-label, randomised controlled trial in 81 hospital-based pulmonary hypertension centres in 22 countries. The study enrolled patients aged 18-75 years with symptomatic PAH at intermediate risk of 1-year mortality (based on the European Society for Cardiology-European Respiratory Society guideline thresholds for WHO functional class and 6-min walk distance [6MWD]) who were receiving treatment with a PDE5i with or without an endothelin receptor antagonist for at least 6 weeks before randomisation. Patients were excluded if they had been previously treated with riociguat, had used prostacyclin analogues or prostacyclin receptor agonists within 30 days before randomisation, had clinically significant restrictive or obstructive parenchymal lung disease, or had left heart disease. Patients were randomly assigned (1:1) to remain on PDE5i treatment (oral sildenafil [≥60 mg per day] or oral tadalafil [20-40 mg per day]; the PDE5i group) or to switch to oral riociguat (up to 2·5 mg three times per day; the riociguat group), using an interactive voice and web response system, stratified by cause of PAH. The primary endpoint was clinical improvement by week 24, defined as an absence of clinical worsening and prespecified improvements in at least two of three variables (6MWD, WHO functional class, and N-terminal prohormone of brain natriuretic peptide), analysed using last observation carried forward in all randomly assigned patients with observed values at baseline and week 24 who received at least one dose of study medication (the full analysis set). Secondary endpoints included clinical worsening events. The trial has been completed and is registered with ClinicalTrials.gov, NCT02891850. FINDINGS: Between Jan 11, 2017, and July 31, 2019, 293 patients were screened, of which 226 patients were randomly assigned to the riociguat group (n=111) or to the PDE5i group (n=115). 211 patients completed the study and 14 patients discontinued (seven in each group). One patient assigned to the PDE5i group did not receive treatment, so 225 patients were included in the safety analysis, and one further patient in the PDE5i group had missing components of the composite primary endpoint at baseline, so 224 patients were included in the full analysis set. The primary endpoint was met by 45 (41%) of 111 patients in the riociguat group and 23 (20%) of 113 patients in the PDE5i group; odds ratio [OR] 2·78 (95% CI 1·53-5·06; p=0·0007). Clinical worsening events occurred in one (1%) of 111 patients in the riociguat group (hospitalisation due to worsening PAH) and 10 (9%) of 114 patients in the PDE5i group (hospitalisation due to worsening PAH [n=9]; disease progression [n=1]; OR 0·10 [0·01-0·73]; p=0·0047). The most frequently occurring adverse events were hypotension (15 [14%]), headache (14 [13%]), and dyspepsia (10 [9%]) in the riociguat group, and headache (eight [7%]), cough (seven [6%]), and upper respiratory tract infection (seven [6%]) in the PDE5i group. Serious adverse events were reported in eight (7%) of 111 patients in the riociguat group and 19 (17%) of 114 patients in the PDE5i group. During the study, four patients died in the PDE5i group, one of them during the safety follow-up period. INTERPRETATION: Switching to riociguat from PDE5i treatment, both of which act via the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate pathway, could be a strategic option for treatment escalation in patients with PAH at intermediate risk of 1-year mortality. FUNDING: Bayer AG, Merck Sharp & Dohme.

Assessment of the REPLACE study composite endpoint in riociguat-treated patients in the PATENT study.

The goal of treatment in patients with pulmonary arterial hypertension is to achieve a low risk status, indicating a favorable long-term outcome. The REPLACE study investigated the efficacy of switching to riociguat in patients with pulmonary arterial hypertension and an insufficient response to phosphodiesterase-5 inhibitors. In this post hoc analysis, we applied the REPLACE composite endpoint of clinical improvement to the placebo-controlled PATENT-1 study of riociguat in pulmonary arterial hypertension and its long-term extension, PATENT-2. Clinical improvement was defined as ≥2 of the following in patients who completed the study without clinical worsening: ≥10% or ≥30 m improvement in 6-minute walking distance; World Health Organization functional class I or II; ≥30% decrease in N-terminal prohormone of brain natriuretic peptide. At PATENT-1 Week 12, patients treated with riociguat were more likely to achieve the composite endpoint vs. placebo (P < 0.0001), with similar results in pretreated (P = 0.0189) and treatment-naïve (P < 0.0001) patients. Achievement of the composite endpoint at Week 12 was associated with a 45% reduction in relative risk of death and a 19% reduction in relative risk of clinical worsening in PATENT-2. Overall, these data suggest that use of the REPLACE composite endpoint in patients with pulmonary arterial hypertension is a valid assessment of response to treatment.

Riociguat improves health-related quality of life for patients with pulmonary arterial hypertension: results from the phase 4 MOTION study.

Pulmonary arterial hypertension (PAH) is characterized by progressive dyspnea and exercise limitation and is associated with reduced health-related quality of life. Few clinical studies have evaluated the primary effects of treatment of PAH from the patient perspective. Here, we present the impact of riociguat on patient-reported outcomes (PROs) in treatment-naïve patients with PAH. MOTION (NCT02191137) was an open-label, phase 4 trial of riociguat monotherapy in treatment-naïve patients with PAH. The primary endpoint was the change in total score from baseline to Week 24 in the Living with Pulmonary Hypertension (LPH) questionnaire. The Short Form-12 Health Survey and Work Limitations Questionnaire 8 were also utilized to assess PROs. Other secondary endpoints included change from baseline in World Health Organization functional class (WHO FC), 6-min walk distance (6MWD), Modified Borg Dyspnea Scale, and safety. At week 24 (n = 66), the mean (standard deviation [SD]) total LPH score was 37.17 (24.61), for a mean (SD) change from baseline of -10.99 (22.51). At last visit, with week 24 imputed, the mean (SD) total score was 40.63 (28.38), for a mean (SD) change from baseline of -5.40 (27.8) (n = 75; P = 0.0484). Improvement in LPH questionnaire total score was observed by week 4 and was maintained through week 24. Improvements were observed in WHO FC, Modified Borg Dyspnea Scale, and accelerometer-measured 6MWD at week 24. Treatment with riociguat had a positive impact on PROs in treatment-naïve patients with PAH and was well tolerated, with a similar safety profile to that observed in placebo-controlled phase 3 trials.

Switching inhaled iloprost formulations in patients with pulmonary arterial hypertension: the VENTASWITCH Trial.

Inhaled iloprost is an effective therapy for patients with pulmonary arterial hypertension (PAH); however, some patients experience extended inhalation times when using the V10 formulation (10.0 µg/mL) to deliver a 5 -µg dose (at mouthpiece) and are at risk of incomplete inhalations and reduced inhalation frequency. VENTASWITCH was an observational, case-crossover study to evaluate inhalation behavior in patients with PAH switched from iloprost V10 to V20 (20.0 µg/mL) formulation for delivering a 5 -µg dose using the I-Neb® AAD® device. Adults with PAH participating in a German Ventavis® (iloprost) patient-support program, who were switched from the V10 to V20 formulation, were enrolled. The co-primary endpoints were mean daily proportion of complete inhalations and mean daily inhalation frequency. The secondary endpoint was mean daily inhalation duration. Data were collected for three months before and after switching. Overall, 63 patients were included. Switching from V10 to V20 resulted in a significant increase in the mean daily proportion of complete inhalations (92% vs. 97%, P < 0.0001) and inhalation frequency (4.6 vs. 4.9 inhalations/day, P = 0.0430), and reduction in mean inhalation duration (11.8 vs. 6.5 min; P < 0.0001). Greater increases in daily proportions of complete inhalations were observed in older patients (≥ 65 vs. < 65 years) and those receiving more (3 vs. < 3) concomitant PAH medications. Switching from V10 to V20 iloprost formulation significantly improved inhalation behavior in patients with PAH and may facilitate improved adherence to therapy.

A detailed view on pacemaker lead parameters remotely transmitted after magnetic resonance.

BACKGROUND: The purpose of this study was to analyze potential influences of magnetic resonance (MR) on the course of automatically device-based assessed lead parameters remotely transmitted in patients who were implanted with MR-conditional permanent pacemakers (PMs) and who had nondiagnostic brain and lumbar spine MR (1.5T) within the ProMRI single center pilot study. METHODS: The ProMRI study evaluated the feasibility of the Evia PMs with Safio S leads (Biotronik SE&Co KG, Berlin, Germany) in the MR environment. All patients were equipped with remote monitoring on the day of MR. Atrial (RA) und ventricular (RV) lead parameters (sensing, pacing capture threshold [PCT], pacing impedance) were automatically assessed and remotely transmitted on a daily or event-triggered basis for 3 months post MR. Remotely transmitted data were normalized for potential differences between at-daytime (in-office) and at-night-time (remotely) assessed parameters using the 1-month follow-up data for each patient. Confidence intervals of continuous data were calculated day-wise with one sample t-tests of post-MR/pre-MR differences, respectively. RESULTS: A total of 2,428 data sets (mean 80 ± 20 per patient) were transmitted. Mean values for the different lead parameters were (RA/RV) 3.3 ± 2.0/14.4 ± 6.9 mV for sensing, 0.65 ± 0.17/0.78 ± 0.23 V/0.4 ms for PCT, and 516 ± 60/607 ± 47 Ω for pacing impedance. No significant differences were found compared with pre-MR measurements. No atrial PCT increases ≥0.5 V compared with pre-MR were observed, and in only one patient the ventricular PCT increased by ≥0.5 V from day 76 post-MR, presumably based on new antiarrhythmic therapy with amiodarone. CONCLUSION: Our analyses of automatically assessed and remotely transmitted PM lead parameters after MR show that sensing amplitudes, PCTs, and pacing impedances are not affected in a clinically relevant way by MR.

A probabilistic model for the carry-over of PCDD/Fs from feed to growing pigs.

When food producing animals are contaminated with PCDD/F congeners, information on the contaminant's concentration in the bodies of the animals at time of slaughter is needed for risk management purposes. We have developed a mathematical model for the kinetics of PCDD/Fs in growing pigs in case of contaminated feed fed for a limited duration of time. This model allows the prediction of concentrations in body fat. It considers absorption fractions of PCDD/Fs, clearance by metabolism, dilution by growth and excretion through fecal fat. The model parameters were calibrated by fitting the model to experimental data. On the basis of this toxicokinetic model a probabilistic model has been constructed. The probabilistic model handles the parameters with appropriate probability distributions and Monte-Carlo simulation technique, providing for realistic situations with many animals and a range of contaminations and feeding intervals. We applied the new model to describe the German dioxin incident of winter 2010/2011 and discuss its viability as decision tool. The approach demonstrated here is a showcase how a risk assessment in the case of contaminated feeding can be performed.

PPINGUIN: Peptide Profiling Guided Identification of Proteins improves quantitation of iTRAQ ratios.

BACKGROUND: Recent development of novel technologies paved the way for quantitative proteomics. One of the most important among them is iTRAQ, employing isobaric tags for relative or absolute quantitation. Despite large progress in technology development, still many challenges remain for derivation and interpretation of quantitative results. One of these challenges is the consistent assignment of peptides to proteins. RESULTS: We have developed Peptide Profiling Guided Identification of Proteins (PPINGUIN), a statistical analysis workflow for iTRAQ data addressing the problem of ambiguous peptide quantitations. Motivated by the assumption that peptides uniquely derived from the same protein are correlated, our method employs clustering as a very early step in data processing prior to protein inference. Our method increases experimental reproducibility and decreases variability of quantitations of peptides assigned to the same protein. Giving further support to our method, application to a type 2 diabetes dataset identifies a list of protein candidates that is in very good agreement with previously performed transcriptomics meta analysis. Making use of quantitative properties of signal patterns identified, PPINGUIN can reveal new isoform candidates. CONCLUSIONS: Regarding the increasing importance of quantitative proteomics we think that this method will be useful in practical applications like model fitting or functional enrichment analysis. We recommend to use this method if quantitation is a major objective of research.

Biomarker discovery and redundancy reduction towards classification using a multi-factorial MALDI-TOF MS T2DM mouse model dataset.

BACKGROUND: Diabetes like many diseases and biological processes is not mono-causal. On the one hand multi-factorial studies with complex experimental design are required for its comprehensive analysis. On the other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics. RESULTS: We present a comprehensive work-flow tailored for analyzing complex data including data from multi-factorial studies. The developed approach aims at revealing effects caused by a distinct combination of experimental factors, in our case genotype and diet. Applying the developed work-flow to the analysis of an established polygenic mouse model for diet-induced type 2 diabetes, we found peptides with significant fold changes exclusively for the combination of a particular strain and diet. Exploitation of redundancy enables the visualization of peptide correlation and provides a natural way of feature selection for classification and prediction. Classification based on the features selected using our approach performs similar to classifications based on more complex feature selection methods. CONCLUSIONS: The combination of ANOVA and redundancy exploitation allows for identification of biomarker candidates in multi-dimensional MALDI-TOF MS profiling studies with complex experimental design. With respect to feature selection our method provides a fast and intuitive alternative to global optimization strategies with comparable performance. The method is implemented in R and the scripts are available by contacting the corresponding author.

A meta-analysis of QTL for diabetes-related traits in rodents.

Crossbreeding studies in rodents have identified numerous quantitative trait loci (QTL) that are linked to diabetes-related component traits. To identify genetic consensus regions implicated in insulin action and glucose homeostasis, we have performed a meta-analysis of genomewide linkage scans for diabetes-related traits. From a total of 43 published genomewide scans we assembled a nonredundant collection of 153 QTL for glucose levels, insulin levels, and glucose tolerance. Collectively, these studies include data from 48 different parental strains and >11,000 individual animals. The results of the studies were analyzed by the truncated product method (TPM). The analysis revealed significant evidence for linkage of glucose levels, insulin levels, and glucose tolerance to 27 different segments of the mouse genome. The most prominent consensus regions [localized to chromosomes 2, 4, 7, 9, 11, 13, and 19; logarithm of odds (LOD) scores 10.5-17.4] cover approximately 11% of the mouse genome and collectively contain the peak markers for 47 QTL. Approximately half of these genomic segments also show significant linkage to body weight and adiposity, indicating the presence of multiple obesity-dependent and -independent consensus regions for diabetes-related traits. At least 84 human genetic markers from genomewide scans and >80 candidate genes from human and rodent studies map into the mouse consensus regions for diabetes-related traits, indicating a substantial overlap between the species. Our results provide guidance for the identification of novel candidate genes and demonstrate the presence of numerous distinct consensus QTL regions with highly significant LOD scores that control glucose homeostasis. An interactive physical map of the QTL is available online at http://www.diabesitygenes.org.

Systematic interpretation of cyclic nucleotide binding studies using KinetXBase.

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.

Differential binding studies applying functional protein microarrays and surface plasmon resonance.

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ.

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are L-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to D-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPgammaS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels.