Reconstruction of cell-specific models capturing the influence of metabolism on DNA methylation in cancer.

The imbalance of epigenetic regulatory mechanisms such as DNA methylation, which can promote aberrant gene expression profiles without affecting the DNA sequence, may cause the deregulation of signaling, regulatory, and metabolic processes, contributing to a cancerous phenotype. Since some metabolites are substrates and cofactors of epigenetic regulators, their availability can be affected by characteristic cancer cell metabolic shifts, feeding cancer onset and progression through epigenetic deregulation. Hence, there is a need to study the influence of cancer metabolic reprogramming in DNA methylation to design new effective treatments. In this study, a generic Genome-Scale Metabolic Model (GSMM) of a human cell, integrating DNA methylation or demethylation reactions, was obtained and used for the reconstruction of Genome-Scale Metabolic Models enhanced with Enzymatic Constraints using Kinetic and Omics data (GECKOs) of 31 cancer cell lines. Furthermore, cell-line-specific DNA methylation levels were included in the models, as coefficients of a DNA composition pseudo-reaction, to depict the influence of metabolism over global DNA methylation in each of the cancer cell lines. Flux simulations demonstrated the ability of these models to provide simulated fluxes of exchange reactions similar to the equivalent experimentally measured uptake/secretion rates and to make good functional predictions. In addition, simulations found metabolic pathways, reactions and enzymes directly or inversely associated with the gene promoter methylation. Two potential candidates for targeted cancer epigenetic therapy were identified.

Integration of Stemness Gene Signatures Reveals Core Functional Modules of Stem Cells and Potential Novel Stemness Genes.

Stem cells encompass a variety of different cell types which converge on the dual capacity to self-renew and differentiate into one or more lineages. These characteristic features are key for the involvement of stem cells in crucial biological processes such as development and ageing. To decipher their underlying genetic substrate, it is important to identify so-called stemness genes that are common to different stem cell types and are consistently identified across different studies. In this meta-analysis, 21 individual stemness signatures for humans and another 21 for mice, obtained from a variety of stem cell types and experimental techniques, were compared. Although we observed biological and experimental variability, a highly significant overlap between gene signatures was identified. This enabled us to define integrated stemness signatures (ISSs) comprised of genes frequently occurring among individual stemness signatures. Such integrated signatures help to exclude false positives that can compromise individual studies and can provide a more robust basis for investigation. To gain further insights into the relevance of ISSs, their genes were functionally annotated and connected within a molecular interaction network. Most importantly, the present analysis points to the potential roles of several less well-studied genes in stemness and thus provides promising candidates for further experimental validation.

Extracellular vesicles enriched with an endothelial cell pro-survival microRNA affects skin tissue regeneration.

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

Reconstruction of tissue-specific genome-scale metabolic models for human cancer stem cells.

Cancer Stem Cells (CSCs) contribute to cancer aggressiveness, metastasis, chemo/radio-therapy resistance, and tumor recurrence. Recent studies emphasized the importance of metabolic reprogramming of CSCs for the maintenance and progression of the cancer phenotype through both the fulfillment of the energetic requirements and the supply of substrates fundamental for fast-cell growth, as well as through metabolite-induced epigenetic regulation. Therefore, it is of paramount importance to develop therapeutic strategies tailored to target the metabolism of CSCs. In this work, we built computational Genome-Scale Metabolic Models (GSMMs) for CSCs of different tissues. Flux simulations were then used to predict metabolic phenotypes, identify potential therapeutic targets, and spot already-known Transcription Factors (TFs), miRNAs and antimetabolites that could be used as part of drug repurposing strategies against cancer. Results were in accordance with experimental evidence, provided insights of new metabolic mechanisms for already known agents, and allowed for the identification of potential new targets and compounds that could be interesting for further in vitro and in vivo validation.

High-throughput identification of small molecules that affect human embryonic vascular development.

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

StemChecker: a web-based tool to discover and explore stemness signatures in gene sets.

Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu.

The unfolded protein response and its potential role in Huntington's disease elucidated by a systems biology approach.

Huntington ´s disease (HD) is a progressive, neurodegenerative disease with a fatal outcome. Although the disease-causing gene (huntingtin) has been known for over 20 years, the exact mechanisms leading to neuronal cell death are still controversial. One potential mechanism contributing to the massive loss of neurons observed in the brain of HD patients could be the unfolded protein response (UPR) activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, persistent ER stress and an activated UPR can also cause apoptotic cell death. Although different studies have indicated a role for the UPR in HD, the evidence remains inconclusive. Here, we present extensive bioinformatic analyses that revealed UPR activation in different experimental HD models based on transcriptomic data. Accordingly, we have identified 53 genes, including RAB5A, HMGB1, CTNNB1, DNM1, TUBB, TSG101, EEF2, DYNC1H1, SLC12A5, ATG5, AKT1, CASP7 and SYVN1 that provide a potential link between UPR and HD. To further elucidate the potential role of UPR as a disease-relevant process, we examined its connection to apoptosis based on molecular interaction data, and identified a set of 40 genes including ADD1, HSP90B1, IKBKB, IKBKG, RPS3A and LMNB1, which seem to be at the crossroads between these two important cellular processes. Remarkably, we also found strong correlation of UPR gene expression with the length of the polyglutamine tract of Huntingtin, which is a critical determinant of age of disease onset in human HD patients pointing to the UPR as a promising target for therapeutic intervention. The study is complemented by a newly developed web-portal called UPR-HD (http://uprhd.sysbiolab.eu) that enables visualization and interactive analysis of UPR-associated gene expression across various HD models.

Inclusion complexes of ionic liquids and cyclodextrins: are they formed in the gas phase?

The interaction of imidazolium-based ionic liquids with α- and ß-cyclodextrins was investigated by electrospray ionization mass spectrometry with variable collision induced dissociation energy and quantum chemical gas-phase calculations. The center-of-mass energy at which 50% of a precursor ion decomposes (Ecm,1/2) was determined for the isolated [cyclodextrin + cation](+) or [cyclodextrin + anion](-) adduct ions of imidazolium-based ionic liquids with different alkyl chain lengths combined with a large set of anions, such as chloride, bromide, bis(trifluoromethylsulfonyl)imide, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate, dicyanamide, and hydrogensulfate. Moreover, both symmetric and asymmetric imidazolium cationic cores were evaluated. The relative interaction energies in the adduct ions were interpreted in terms of the influence of cation/anion structures and their inherent properties, such as hydrophobicity and hydrogen bond accepting ability, in the complexation process with the cyclodextrins. The trends observed in the mass spectral data together with quantum-chemical calculations suggest that in the gas phase, cations and anions will preferentially interact with the lower or upper rim of the cyclodextrin, respectively, as opposed to what has been reported in condensed phase where the formation of an inclusion complex between ionic liquid and cyclodextrin is assumed.