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.
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.
PurposeEphedra alata (E. alata) is perennial tough shrub plant that grows in Palestine and other regions. It is used often in folk's medicine for the treatment of various diseases. In this project, E. alata extract was tested for its ability to improve wound and burn healing.MethodsAn aqueous extract of E. alata was prepared and underwent several phytochemical analyses for the presence of the major classes of phytochemical compounds. After that, a polyethylene glycol-based ointment containing the extract of E. alata was prepared and its wound and burn healing activities were tested in-vivo using an animal model for deep wound and full thickness skin burn. The effect was compared against a placebo ointment. Skin biopsies were evaluated by a blinded clinical histopathologist, in addition to digital analysis.ResultsPhytochemical analysis demonstrated the presence of the major classes of phytochemical compounds in the prepared extract including flavonoids, alkaloids, phytosteroids, phenolic compounds, volatile oils and tannins. As compared to placebo ointment, E. alata ointment significantly improved the healing of the wound ulcers, whereas it showed no advantage on the quality of the healing of burn ulcers.ConclusionE. alata extract is rich in phytochemical compounds and can improve wound healing when applied topically.
Cardiac remodeling (cardiac hypertrophy and fibrosis) is a hallmark of heart failure (HF). It can be induced by the abnormal elevation of several endogenous factors including angiotensin II (Ang II), which is generated from its precursor angiotensin I (Ang I) by the action of angiotensin‐converting enzyme. The inhibition of this enzyme or the blockade of the Ang II receptors demonstrated a high clinical value against the progression of HF. Ang I and Ang II may also be converted into angiotensin 1‐7 (Ang 1‐7) and angiotensin 1‐9 (Ang 1‐9), respectively, by the action of angiotensin‐converting enzyme 2. Both derivatives demonstrated a promising anticardiac remodeling activity especially against the detrimental effects of Ang II. This manuscript thoroughly reviews the available in vitro and in vivo data on Ang 1‐7 and Ang 1‐9 in the context of the treatment of HF and discusses the associated molecular mechanisms and the trials to clinically utilize Ang 1‐7 mimetics for the treatment of that disease.
Single walled carbon nanotubes (SWCNT) are currently under intensive investigation by many labs all over the world for being promising candidates for cancer chemotherapy delivery. On the other hand, combretastatin A4 (CA4) is an anticancer drug that induces cell apoptosis by inhibiting tubulin polymerization. However, it has the disadvantage of low water solubility and the non-selective targeting. Therefore, we aim to create nano-drug from the functionalization of SWCNT covalently with CA4 through click reaction in the presence of tetraethylene glycol linker in order to improve its dispersibility. Scanning electron microscopy and transmission electron microscopy showed good dispersibility of the functionalized SWCNT with diameters of 5-15 nm. Moreover, thermogravometric analysis showed that the efficiency of SWCNT functionalization was around 45%. The in vitro release profile of CA4 at physiological conditions showed that approximately 90% of the loaded drug was released over 50 h. After that MTS test was used to determine the suitable concentration range for the in vitro investigation of the SWCNT-CA4. After that the cytotoxic activity of the SWCNT-CA4 was evaluated by flow cytometry using annexin V/propidium iodide (PI) test. In comparison with free CA4, SWCNT-CA4 treatment demonstrated a significant increase in necrotic cells (around 50%) at the expense of the proportion of the apoptotic cells. Moreover, cell cycle PI test demonstrated that free CA4 and SWCNT-CA4 caused G2/M arrest. However with CA4 treatment higher proportion of cells were in the S-phase while with SWCNT-CA4 treatment greater proportion of cells appeared to be in the G1-phase. Taken together, the provided data suggest that the novel SWCNT-CA4 has a significant anticancer activity that might be superior to that of free CA4.
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