Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept

One fungus, which genes?: development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation elongation factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail

Loop Quantum Cosmology

Quantum gravity is expected to be necessary in order to understand situations where classical general relativity breaks down. In particular in cosmology one has to deal with initial singularities, i.e. the fact that the backward evolution of a classical space-time inevitably comes to an end after a finite amount of proper time. This presents a breakdown of the classical picture and requires an extended theory for a meaningful description. Since small length scales and high curvatures are involved, quantum effects must play a role. Not only the singularity itself but also the surrounding space-time is then modified. One particular realization is loop quantum cosmology, an application of loop quantum gravity to homogeneous systems, which removes classical singularities. Its implications can be studied at different levels. Main effects are introduced into effective classical equations which allow to avoid interpretational problems of quantum theory. They give rise to new kinds of early universe phenomenology with applications to inflation and cyclic models. To resolve classical singularities and to understand the structure of geometry around them, the quantum description is necessary. Classical evolution is then replaced by a difference equation for a wave function which allows to extend space-time beyond classical singularities. One main question is how these homogeneous scenarios are related to full loop quantum gravity, which can be dealt with at the level of distributional symmetric states. Finally, the new structure of space-time arising in loop quantum gravity and its application to cosmology sheds new light on more general issues such as time.Comment: 104 pages, 10 figures; online version, containing 6 movies, available at http://relativity.livingreviews.org/Articles/lrr-2005-11

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42\ub74% vs 44\ub72%; absolute difference \u20131\ub769 [\u20139\ub758 to 6\ub711] p=0\ub767; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5\u20138] vs 6 [5\u20138] cm H2O; p=0\ub70011). ICU mortality was higher in MICs than in HICs (30\ub75% vs 19\ub79%; p=0\ub70004; adjusted effect 16\ub741% [95% CI 9\ub752\u201323\ub752]; p<0\ub70001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0\ub780 [95% CI 0\ub775\u20130\ub786]; p<0\ub70001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status. Funding: No funding

Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics

The Phenotype MicroArray (OmniLog® PM) system is able to simultaneously capture a large number of phenotypes by recording an organism's respiration over time on distinct substrates. This technique targets the object of natural selection itself, the phenotype, whereas previously addressed '-omics' techniques merely study components that finally contribute to it. The recording of respiration over time, however, adds a longitudinal dimension to the data. To optimally exploit this information, it must be extracted from the shapes of the recorded curves and displayed in analogy to conventional growth curves.The free software environment R was explored for both visualizing and fitting of PM respiration curves. Approaches using either a model fit (and commonly applied growth models) or a smoothing spline were evaluated. Their reliability in inferring curve parameters and confidence intervals was compared to the native OmniLog® PM analysis software. We consider the post-processing of the estimated parameters, the optimal classification of curve shapes and the detection of significant differences between them, as well as practically relevant questions such as detecting the impact of cultivation times and the minimum required number of experimental repeats.We provide a comprehensive framework for data visualization and parameter estimation according to user choices. A flexible graphical representation strategy for displaying the results is proposed, including 95% confidence intervals for the estimated parameters. The spline approach is less prone to irregular curve shapes than fitting any of the considered models or using the native PM software for calculating both point estimates and confidence intervals. These can serve as a starting point for the automated post-processing of PM data, providing much more information than the strict dichotomization into positive and negative reactions. Our results form the basis for a freely available R package for the analysis of PM data

Varying constants, Gravitation and Cosmology

Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. It is thus of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We thus detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, Solar system observations, meteorites dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describe the main theoretical frameworks in which the low-energy constants may actually be varying and we focus on the unification mechanisms and the relations between the variation of different constants. To finish, we discuss the more speculative possibility of understanding their numerical values and the apparent fine-tuning that they confront us with.Comment: 145 pages, 10 figures, Review for Living Reviews in Relativit

DataSheet1_Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.docx

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.</p

DataSheet5_Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.docx

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.</p

DataSheet3_Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.docx

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.</p