In this work we aim to show how Genome Scale Metabolic Models (GSMMs) can be used as tools for drug design. By comparing the chemical structures of human metabolites (obtained using their KEGG indexes) and the compounds contained in the DrugBank database, we have observed that compounds showing Tanimoto scores higher than 0.9 with a metabolite, are 29.5 times more likely to bind the enzymes metabolizing the considered metabolite, than ligands chosen randomly. By using RNA-seq data to constrain a human GSMM it is possible to obtain an estimation of its distribution of metabolic fluxes and to quantify the effects of restraining the rate of chosen metabolic reactions (for example using a drug that inhibits the enzymes catalyzing the mentioned reactions). This method allowed us to predict the differential effects of lipoamide analogs on the proliferation of MCF7 (a breast cancer cell line) and ASM (airway smooth muscle) cells respectively. These differential effects were confirmed experimentally, which provides a proof of concept of how human GSMMs could be used to find therapeutic windows against cancer. By using RNA-seq data of 34 different cancer cell lines and 26 healthy tissues, we assessed the putative anticancer effects of the compounds in DrugBank which are structurally similar to human metabolites. Among other results it was predicted that the mevalonate pathway might constitute a good therapeutic window against cancer proliferation, due to the fact that most cancer cell lines do not express the cholesterol transporter NPC1L1 and the lipoprotein lipase LPL, which makes them rely on the mevalonate pathway to obtain cholesterol.

Results of our experiments indicate that ionic strength of the incubation medium influence the selectivity, the medium temperature and the assay conditions impact the kinetics of efflux. The lower accumulated amount and the weaker fluorescence of Et registered in slightly acidic medium indicate that ΔΨ plays a role in the accumulation of this indicator cation. The bound amount of Et to the de-energized or permeabilized cells considerably varies depending on the conditions and methods of de-energization or permeabilization of cells. Tris/EDTA permeabilization of the cells does not inhibit the efflux.

Both cytosolic fatty acid synthesis (FAS) and mitochondrial fatty acid oxidation (FAO) have been shown to play a role in the survival and proliferation of cancer cells. This study aimed to confirm experimentally whether FAS and FAO coexist in breast cancer cells (BCC). By feeding cells with 13C-labeled glutamine and measuring labeling patterns of TCA intermediates, it was possible to show that part of the cytosolic acetyl-CoA used in lipid synthesis is also fed back into the mitochondrion via fatty acid degradation. This results in the transfer of reductive potential from the cytosol (in the form of NADPH) to the mitochondrion (in the form of NADH and FADH2). The hypothesized mechanism was further confirmed by blocking FAS and FAO with siRNAs. Exposure to staurosporine (which induces ROS production) resulted in the disruption of simultaneous FAS and FAO, which could be explained by NADPH depletion.

Engineering of sophisticated synthetic 3D scaffolds that allow controlling behaviour and location of the cells requires advanced micro/nano-fabrication techniques. Ultrafast laser micro-machining employing a 1030-nm wavelength Yb:KGW femtosecond laser and a micro-fabrication workstation for micro-machining of commercially available 12.7 and 25.4 μm thickness polyimide (PI) film was applied. Mechanical properties of the fabricated scaffolds, i.e. arrays of differently spaced holes, were examined via custom-built uniaxial micro-tensile testing and finite element method simulations. We demonstrate that experimental micro-tensile testing results could be numerically simulated and explained by two-material model, assuming that 2-6 μm width rings around the holes possessed up to five times higher Young's modulus and yield stress compared with the rest of the laser intacted PI film areas of 'dog-bone'-shaped specimens. That was attributed to material modification around the micro-machined holes in the vicinity of the position of the focused laser beam track during trepanning drilling. We demonstrate that virgin PI films provide a suitable environment for the mobility, proliferation and intercellular communication of human bone marrow mesenchymal stem cells, and discuss how cell behaviour varies on the micro-machined PI films with holes of different diameters (3.1, 8.4 and 16.7 μm) and hole spacing (30, 35, 40 and 45 μm). We conclude that the holes of 3.1 μm diameter were sufficient for metabolic and genetic communication through membranous tunneling tubes between cells residing on the opposite sides of PI film, but prevented the trans-migration of cells through the holes. Copyright © 2016 John Wiley & Sons, Ltd.

A comparative analysis between cancer cell lines and healthy dividing cells was performed using data (289 microarrays and 50 RNA-seq samples) from 100 different cancer cell lines and 6 types of healthy stem cells. The analysis revealed two large-scale transcriptional events that characterize cancer cell lines. The first event was a large-scale up-regulation pattern associated to epithelial-mesenchymal transition, putatively driven by the interplay of the SP1 transcription factor and the canonical Wnt signaling pathway; the second event was the failure to overexpress a diverse set of genes coding membrane and extracellular proteins. This failure is putatively caused by a lack of activity of the AP-1 complex. It was also shown that the epithelial-mesenchymal transition was associated with the up-regulation of 5 enzymes involved in the degradation of branched chain amino acids. The suitability of silencing one of this enzymes (branched chain amino acid transaminase 2; BCAT2) with therapeutic effects was tested experimentally on the breast cancer cell line MCF-7 and primary cell culture of breast tumor (BCC), leading to lower cell proliferation. The silencing of BCAT2 did not have any significant effect on ASM and MCF10A cells, which were used as models of healthy dividing cells.