Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (C max) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC 10) yielded better metrics than higher toxicity thresholds (EC 50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of C max were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC 10 and C max as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

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BackgroundImpaired wound healing is commonly associated with many health problems, including diabetes, bedsores and extensive burns. In such cases, healing often takes a long time, which subjects patients to various complications. This study aims to investigate whether single-wall or multi-wall carbon nanotubes complexed with chitosan hydrogel can improve wound healing.Materials and methodsInitially, the effects of the complexes on the viability and functionality of fibroblasts were investigated in engineered connective tissues. Then, their activity on wound healing was investigated in a mouse model with induced full-thickness wounds, in which the wounds were treated daily with these complexes. Finally, the effect of the complexes on collagen deposition by fibroblasts was investigated in vitro.ResultsThe engineered connective tissue studies showed that fibroblasts were viable in the presence of the complexes and were still able to effectively organize and contract the extracellular matrix. In vivo data showed that both types of complexes improved the re-epithelialization of the healing wounds; however, they also increased the percentage of wounds with higher fibrosis. In particular, the chitosan-multi-wall carbon nanotube complex significantly enhanced the extensiveness of this fibrosis, which is in line with in vitro data showing a concentration-dependent enhancement of collage deposition by these complexes. These observations were associated with an increase in inflammatory signs in the wound bed.ConclusionSingle-wall and multi-wall carbon nanotubes complexed with chitosan improved the re-epithelialization of wounds, but an increase in fibrosis was detected.

The ongoing spread of multi-drug-resistant bacteria over the past few decades necessitates collateral efforts to develop new classes of antibacterial agents with different mechanisms of action. The utilization of graphene nanosheets has recently gained attention with this respect. Herein, we have synthesized and tested the antibacterial activity of an array of graphene materials covalently functionalized with hydroxyl-, amine-, or carboxyl-containing groups. Fourier transform infrared spectroscopy and transmission electron microscopy confirmed successful functionalization of the few-layer graphene (FLG). The percentage of weight loss was measured by thermogravimetric analysis, which was found to be 22%, 23%, and 37% for FLG-TEG-OH, FLG-NH2, and FLG-DEG-COOH, respectively. In comparison with pristine graphene sheets, the functionalized few-layer graphene (f-FLG) materials gained an adequate dispersibility in water as confirmed by ζ potential analysis. Moreover, there was a significant improvement in the antibacterial activity against Staphylococcus aureus and Escherichia coli, where all f-FLG compounds were able to suppress bacterial growth, with a complete suppression achieved by FLG-DEG-COOH. The minimum inhibitory concentration (MIC) was 250 μg mL–1 for both FLG-TEG-OH and FLG-NH2, while it was 125 μg mL–1 for FLG-DEG-COOH. The glutathione oxidation test demonstrated an oxidative stress activity by all f-FLG compounds. However, FLG-DEG-COOH demonstrated the highest reduction in glutathione activity. FLG-DEG-COOH and FLG-TEG-OH showed adequate biocompatibility and hemocompatibility. The chemical functionalization of graphene might be a step toward the foundation of an effective class of antimicrobial agents.

IntroductionRhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.ResultsDownregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.ConclusionRhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.