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A platform technology for the automated reaction control in magnetizable micro-fluidic droplets
(2014)
Raphaëlle Luisier
; Harri Lempiäinen
; Nina Scherbichler
; Albert Braeuning
; Miriam Geissler
; Valerie Dubost
; Arne Müller
; Nico Scheer
; Salah-Dine Chibout
; Hisanori Hara
; Frank Picard
; Diethilde Theil
; Philippe Couttet
; Antonio Vitobello
; Olivier Grenet
; Bettina Grasl-Kraupp
; Heidrung Ellinger-Ziegelbauer
; John P. Thomson
; Richard R. Meehan
; Clifford R. Elcombe
; Colin J. Henderson
; C. Roland Wolf
; Michael Schwarz
; Pierre Moulin
; Remi Terranova
; Jonathan G. Moggs
The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CARᴷᴼ-PXRᴷᴼ), double humanized CAR and PXR (CARʰ-PXRʰ), and wild-type C57BL/6 mice. Wild-type and CARʰ-PXRʰ mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CARᴷᴼ-PXRᴷᴼ mouse livers and largely reversible in wild-type and CARʰ-PXRʰ mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CARʰ-PXRʰ mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.
Laurent Salpati
; Xiaoyan Chu
; Liangfu Chen
; Bhagwat Prasad
; Shannon Dallas
; Raymond Evers
; Donna Mamaril-Fishman
; Ethan G. Geier
; Jonathan Kehler
; Jeevan Kunta
; Mario Mezler
; Loic Laplanche
; Jodie Pang
; Matthew G. Soars
; Jashvant D. Unadkat
; Robert A.B. van Waterschoot
; Jocelyn Yabut
; Alfred H. Schinkel
; Nico Scheer
; Anja Rode
Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography–tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.
Nico Scheer
; Lesley A. Mclaughlin
; Anja Rode
; Alastair Kenneth MacLeod
; Colin J. Henderson
; Roland C. Wolf
In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.
1. Drug metabolizing enzymes and transporters play important roles in the absorption, metabolism, tissue distribution and excretion of various compounds and their metabolites and thus can significantly affect their efficacy and safety. Furthermore, they can be involved in drug–drug interactions which can result in adverse responses, life-threatening toxicity or impaired efficacy. Significant species differences in the interaction of compounds with drug metabolizing enzymes and transporters have been described.
2. In order to overcome the limitation of animal models in accurately predicting human responses, a large variety of mouse models humanized for drug metabolizing enzymes and to a lesser extent drug transporters have been created.
3. This review summarizes the literature describing these mouse models and their key applications in studying the role of drug metabolizing enzymes and transporters in drug bioavailability, tissue distribution, clearance and drug–drug interactions as well as in human metabolite testing and risk assessment.
4. Though such humanized mouse models have certain limitations, there is great potential for their use in basic research and for testing and development of new medicines. These limitations and future potentials will be discussed.
Molecular Modeling Approach to the Prediction of Mechanical Properties of Silica-Reinforced Rubbers
(2014)
Recently, we have suggested a nanomechanical model for dissipative loss in filled elastomer networks in the context of the Payne effect. The mechanism is based on a total interfiller particle force exhibiting an intermittent loop, due to the combination of short-range repulsion and dispersion forces with a long-range elastic attraction. The sum of these forces leads, under external strain, to a spontaneous instability of “bonds” between the aggregates in a filler network and attendant energy dissipation. Here, we use molecular dynamics simulations to obtain chemically realistic forces between surface modified silica particles. The latter are combined with the above model to estimate the loss modulus and the low strain storage modulus in elastomers containing the aforementioned filler-compatibilizer systems. The model is compared to experimental dynamic moduli of silica filled rubbers. We find good agreement between the model predictions and the experiments as function of the compatibilizer's molecular structure and its bulk concentration.
Rebecca Schroeter
; Tamara Hoffmann
; Birgit Voigt
; Hanna Meyer
; Monika Bleisteiner
; Jan Muntel
; Britta Jürgen
; Dirk Albrecht
; Dörte Becher
; Michael Lalk
; Stefan Evers
; Johannes Bongaerts
; Karl-Heinz Maurer
; Harald Putzer
; Michael Hecker
; Thomas Schweder
; Erhard Bremer
The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.
Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress
(2014)
Stefan Handtke
; Rebecca Schroeter
; Britta Jürgen
; Karen Methling
; Rabea Schlüter
; Dirk Albrecht
; Sacha A. F. T. van Hijum
; Johannes Bongaerts
; Karl-Heinz Maurer
; Michael Lalk
; Thomas Schweder
; Michael Hecker
; Birgit Voigt
Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.
We present an electromechanically coupled Finite Element model for cardiac tissue. It bases on the mechanical model for cardiac tissue of Hunter et al. that we couple to the McAllister-Noble-Tsien electrophysiological model of purkinje fibre cells. The corresponding system of ordinary differential equations is implemented on the level of the constitutive equations in a geometrically and physically nonlinear version of the so-called edge-based smoothed FEM for plates. Mechanical material parameters are determined from our own pressure-deflection experimental setup. The main purpose of the model is to further examine the experimental results not only on mechanical but also on electrophysiological level down to ion channel gates. Moreover, we present first drug treatment simulations and validate the model with respect to the experiments.