Epithelial-to-mesenchymal transition involves triacylglycerol accumulation in DU145 prostate cancer cells

Epithelial to mesenchymal transition (EMT) is a biological process that plays a crucial role in cancer metastasis. Although studies regarding the EMT mechanisms are usual in terms of gene expression and protein functions, little is known about the involvement of lipids in EMT. In this work, an untargeted lipidomic analysis was performed to reveal which lipids are involved in the EMT process. DU145 prostate cancer cells were treated with TNFα, a well-known EMT inducer. After 6 hours of treatment, a decrease of cell membrane E-cadherin as well as a reduction in its gene expression were observed. Also, the mesenchymal markers Vimentin and Snail were up-regulated, suggesting that EMT started below 6 hours of treatment. Lipid extracts of untreated and TNFα-treated cells at short times were analyzed using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-MS). Multivariate data analysis methods were applied to decipher which lipids presented significant changes after EMT induction. Among the results obtained, a significant increase of twelve unsaturated triacylglycerides (TAGs) was observed. This increase of TAGs was also observed for cells treated with TGFβ (another EMT inducer), suggesting that this feature is a common mechanism in the EMT process. In conclusion, this work reported for the first time a TAG accumulation through EMT induction. These TAG lipids could play a key role in providing cells with the energy, cell membrane components and signaling lipids necessary to guarantee the enhanced cell migration and proliferation of metastatic cells.This work was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 320737. J.J acknowledges a CSIC JAE-Doc contract cofounded by FSE.Peer reviewe

Resolution of a structural competition involving dimeric G-quadruplex and its C-rich complementary strand

The resolution of the dimeric intermolecular G-quadruplex/duplex competition of the telomeric DNA sequence 5′-TAG GGT TAG GGT-3′ and of its complementary 5′ ACC CTA ACC CTA-3′ is reported. To achieve this goal, melting experiments of both sequences and of the mixtures of these sequences were monitored by molecular absorption, molecular fluorescence and circular dichroism spectroscopies. Molecular fluorescence measurements were carried out using molecular beacons technology, in which the 5′-TAG GGT TAG GGT-3′ sequence was labelled with a fluorophore and a quencher at the ends of the strand. Mathematical analysis of experimental spectroscopic data was performed by means of multivariate curve resolution, allowing the calculation of concentration profiles and pure spectra of all resolved structures (dimeric antiparallel and parallel G-quadruplexes, Watson–Crick duplex and single strands) present in solution. Our results show that parallel G-quadruplex is more stable than antiparallel G-quadruplex. When the complementary C-rich strand is present, a mixture of both G-quadruplex structures and Watson–Crick duplex is observed, the duplex being the major species. In addition to melting temperatures, equilibrium constants for the parallel/antiparallel G-quadruplex equilibrium and for the G-quadruplex/duplex equilibrium were determined from the concentration profiles

Study of the interaction between the G-quadruplex-forming thrombin-binding aptamer and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate

The G-quadruplex DNA structure has been suggested to be a potential target for anticancer therapies. Therefore, there is increasing interest in the development of drugs that could modulate the stability of G-quadruplex structures. In the current work, the interaction between the thrombin-binding aptamer (TBA, 5′-GGT TGG TGT GGT TGG-3′), which can form an intramolecular G-quadruplex structure, and the porphyrin 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate (TmPyP4) was studied. The application of a high-performance liquid chromatography-photodiode array (HPLC-PDA) detector-based method to study this kind of interaction was tested. Molecular absorption data recorded along the chromatographic runs were analyzed by means of multivariate data analysis methods. Moreover, biospecific interaction analysis (BIA) by surface plasmon resonance (SPR) and melting and mole ratio experiments monitored by UV-visible molecular absorption and circular dichroism spectroscopies, were applied to confirm and expand the chromatographic studies. The results showed the formation of an interaction complex with a stoichiometry 1:1 (TmPyP4/TBA) and logarithm of the equilibrium constant equal to 5.7 ± 0.2. Melting and circular dichroism data reflected that the initial G-quadruplex structure of TBA is stabilized in the interaction complex, being slightly distorted by the presence of the ligand. © 2008 Elsevier Inc. All rights reserved.This research was supported by the grants CTQ2006-15052-C02-01/BQU, CTQ2007- 28940-E/BQU, and BFU2007-63287/BMC from the Spanish Ministerio de Educación y Ciencia.Peer reviewe

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background: G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods: Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results: We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions: Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance: The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. © 2012 Elsevier B.V.This research was supported by the Spanish Ministerio de Ciencia e Innovación (grant numbers CTQ2009-11572 and CTQ2010-20541-C03-01), and the Generalitat de Catalunya (grant numbers 2009-SGR-45 and 2009- SGR-208).Peer reviewe

Implementació d'un nou sistema de precàrrega de biguetes tipus noubau: el fusible mecànic

El present document és un estudi del comportament i el desenvolupament d'un fusible mecànic per al sistema NOU\BAU. Aquest estudi és una part important del projecte de la implantació de fusibles mecànics en el procés de muntatge del sistema de reforç de biguetes NOU\BAU. La realització d'aquest estudi és important per a desenvolupar i després conèixer el comportament d'un nou sistema d'entrada en càrrega de les bigues mitjançant un element fusible. El present estudi conté un breu resum històric del reforç de biguetes a nivell industrial; una explicació del sistema NOU\BAU; una revisió de l'estat de l'art que ha portat a desenvolupar aquest nou fusible i tot el treball que s'ha fet, tant a nivell de laboratori com a nivell conceptual. També conté les conclusions de cada assaig que han guiat el rumb de les proves al laboratori i, per últim, una revisió general dels resultats obtinguts i les conclusions globals de tot l'estudi. Els resultats d'aquest estudi passen a formar part del desenvolupament de R+D de Sistemes de Reforç Actiu, S.L., empresa col·laboradora que és on s'ha realitzat l'estudi

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. Graphical abstract Protonation pushes away TMPyP4 molecules from the loops in G-quadruplex structures. The interaction of TMPyP4 porphyrin with the G-quadruplex structure formed by a guanine-rich sequence in the promoter region of c-kit gene was studied. Up to three ligand molecules may be bound to the G-quadruplex structure. Protonation at the loops induces the release of one TMPyP4 molecule

Modeling and subtleties of K-Ras and Calmodulin interaction

K-Ras, one of the most common small GTPases of the cell, still presents many riddles, despite the intense efforts to unveil its mysteries. Such is the case of its interaction with Calmodulin, a small acidic protein known for its role as a calcium ion sensor. Although the interaction between these two proteins and its biological implications have been widely studied, a model of their interaction has not been performed. In the present work we analyse this intriguing interaction by computational means. To do so, both conventional molecular dynamics and scaled molecular dynamics have been used. Our simulations suggest a model in which Calmodulin would interact with both the hypervariable region and the globular domain of K-Ras, using a lobe to interact with each of them. According to the presented model, the interface of helixes α4 and α5 of the globular domain of K-Ras would be relevant for the interaction with a lobe of Calmodulin. These results were also obtained when bringing the proteins together in a step wise manner with the umbrella sampling methodology. The computational results have been validated using SPR to determine the relevance of certain residues. Our results demonstrate that, when mutating residues of the α4-α5 interface described to be relevant for the interaction with Calmodulin, the interaction of the globular domain of K-Ras with Calmodulin diminishes. However, it is to be considered that our simulations indicate that the bulk of the interaction would fall on the hypervariable region of KRas, as many more interactions are identified in said region. All in all our simulations present a suitable model in which K-Ras could interact with Calmodulin at membrane level using both its globular domain and its hypervariable region to stablish an interaction that leads to an altered signalling

Vibrational spectroscopic image analysis of biological material using multivariate curve resolution - alternating least squares

Multivariate data analysis techniques are ideal to decrypt chemical differences between anatomical features or tissue areas in hyperspectral images of biological samples. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and un-mixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually less than 30 - 60 min/image) and are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or single band intensity heap maps. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) are possible

Toward understanding calmodulin plasticity by molecular dynamics

Aim: Calmodulin interacts in many different ways with its ligands. We aim to shed light on its plasticity analysing the changes followed by the linker region and the relative position of the lobes using conventional Molecular Dynamics (cMD), accelerated MD (aMD) and scaled MD (sMD). Materials & Methods: Three different structures of calmodulin are compared, obtaining a total of 2.5 μs of molecular dynamics, which have been analysed using the principal component analysis and clustering methodologies Results: sMD simulations reach conformations that cMD is not able to, without compromising the stability of the protein. On the other hand, aMD requires optimization of the setup parameters to be useful. Conclusion: SMD is useful to study flexible proteins, highlighting those factors that justify its promiscuit