Spectroscopic study of the interaction of actinomycin D with oligonucleotides carrying the central base sequences -XGCY- and -XGGCCY- using multivariate methods

10 pages, 5 figures, 1 table.-- PMID: 17123067 [PubMed].-- Published online Nov 23, 2006.Supporting information (4 pages, 3 suppl. figures) available at: http://www.springerlink.com/content/870650451902431q/MediaObjects/216_2006_946_MOESM1_ESM.docThe interactions of actinomycin D (ACTD) with the oligonucleotides 5′-CAAAGCTTTG-3′, 5′-CATGGC CATG-3′ and 5′-TATGGCCATA-3′ were investigated by means of acid–base titrations and mole-ratio and melting experiments monitored by molecular absorption and circular dichroism (CD) spectroscopies. For each experiment, CD and molecular absorption spectra were recorded at each point in the experiment, and later analyzed via appropriate multivariate data analysis methods. The study of the interactions between these oligonucleotides and ACTD at 25°C showed the formation of an interaction complex with a stoichiometry of 1:1 (ACTD:duplex) and values for the log(formation constant) of 5.1 ± 0.3, 6.4 ± 0.2, and 5.6 ± 0.2, respectively. An additional interaction complex at higher temperatures was also detected, which might be related to the single-stranded forms of the oligonucleotides.We acknowledge two grants from the Spanish Ministerio de Educación y Ciencia (projects BFU2004-02048/BMC and BQU2003-0191).Peer reviewe

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

1H NMR metabolomic study of auxotrophic starvation in yeast using Multivariate Curve Resolution-Alternating Least Squares for Pathway Analysis

Disruption of specific metabolic pathways constitutes the mode of action of many known toxicants and it is responsible for the adverse phenotypes associated to human genetic defects. Conversely, many industrial applications rely on metabolic alterations of diverse microorganisms, whereas many therapeutic drugs aim to selectively disrupt pathogens' metabolism. In this work we analyzed metabolic changes induced by auxotrophic starvation conditions in yeast in a non-targeted approach, using one-dimensional proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and chemometric analyses. Analysis of the raw spectral datasets showed specific changes linked to the different stages during unrestricted yeast growth, as well as specific changes linked to each of the four tested starvation conditions (L-methionine, L-histidine, L-leucine and uracil). Analysis of changes in concentrations of more than 40 metabolites by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) showed the normal progression of key metabolites during lag, exponential and stationary unrestricted growth phases, while reflecting the metabolic blockage induced by the starvation conditions. In this case, different metabolic intermediates accumulated over time, allowing identification of the different metabolic pathways specifically affected by each gene disruption. This synergy between NMR metabolomics and molecular biology may have clear implications for both genetic diagnostics and drug development. © The Author(s) 2016.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 320737. We also thank Dr. Yolanda Pérez for her helpful recommendations on setting up the acquisition parameters for some of the NMR experiments.Peer reviewe

ROIMCR: a powerful analysis strategy for LC-MS metabolomic datasets

[Background] The analysis of LC-MS metabolomic datasets appears to be a challenging task in a wide range of disciplines since it demands the highly extensive processing of a vast amount of data. Different LC-MS data analysis packages have been developed in the last few years to facilitate this analysis. However, most of these strategies involve chromatographic alignment and peak shaping and often associate each “feature” (i.e., chromatographic peak) with a unique m/z measurement. Thus, the development of an alternative data analysis strategy that is applicable to most types of MS datasets and properly addresses these issues is still a challenge in the metabolomics field.[Results] Here, we present an alternative approach called ROIMCR to: i) filter and compress massive LC-MS datasets while transforming their original structure into a data matrix of features without losing relevant information through the search of regions of interest (ROIs) in the m/z domain and ii) resolve compressed data to identify their contributing pure components without previous alignment or peak shaping by applying a Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) analysis. In this study, the basics of the ROIMCR method are presented in detail and a detailed description of its implementation is also provided. Data were analyzed using the MATLAB (The MathWorks, Inc., www.mathworks.com) programming and computing environment. The application of the ROIMCR methodology is described in detail, with an example of LC-MS data generated in a lipidomic study and with other examples of recent applications.[Conclusions] The methodology presented here combines the benefits of data filtering and compression based on the searching of ROI features, without the loss of spectral accuracy. The method has the benefits of the application of the powerful MCR-ALS data resolution method without the necessity of performing chromatographic peak alignment or modelling. The presented method is a powerful alternative to other existing data analysis approaches that do not use the MCR-ALS method to resolve LC-MS data. The ROIMCR method also represents an improved strategy compared to the direct applications of the MCR-ALS method that use less-powerful data compression strategies such as binning and windowing. Overall, the strategy presented here confirms the usefulness of the ROIMCR chemometrics method for analyzing LC-MS untargeted metabolomics data.The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013) / ERC Grant Agreement n. 320737. The first author acknowledges the Spanish Government (Ministerio de Educación, Cultura y Deporte) for a predoctoral FPU scholarship. The authors acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). Grant support from Generalitat de Catalunya 2017-SGR-753 and Spanish Ministry of Economy, Industry and Competitiveness (project CTQ2015–66254-C2–1-P) is also acknowledged.Peer reviewe

Perfluoroalkylated substances effects in Xenopus laevis A6 kidney epithelial cells determined by ATR-FTIR spectroscopy and chemometric analysis

The effects of four perfluoroalkylated substances (PFASs), namely, perfluorobutanesulfonate (PFBS), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS) and perfluorononanoic acid (PFNA) were assessed in Xenopus laevis A6 kidney epithelial cells by attenuated total reflection Fouriertransform infrared (ATR-FTIR) spectroscopy and chemometric analysis. Principal component analysis-linear discriminant analysis (PCA-LDA) was used to visualize wavenumber-related alterations and ANOVA-simultaneous component analysis (ASCA) allowed data processing considering the underlying experimental design. Both analyses evidenced a higher impact of low-dose PFAS-treatments (10-9 M) on A6 cells forming monolayers, while there was a larger influence of high-dose PFAS-treatments (10-5 M) on A6 cells differentiated into dome structures. The observed dose-response PFAS-induced effects were to some extent related to their cytotoxicity: the EC50-values of most influent PFAS-treatments increased (PFOS<PFNA<PFOA<<PFBS), higherdoses of these chemicals induced a larger impact. Major spectral alterations were mainly attributed to DNA/RNA, secondary protein structure, lipids and fatty acids. Finally, PFOS and PFOA caused a decrease in A6 cell numbers compared to controls, whereas PFBS and PFNA did not significantly change cell population levels. Overall, this work highlights the ability of PFASs to alter A6 cells, whether forming monolayers or differentiated into dome structures, and the potential of PFOS and PFOA to induce cell death

Multivariate Curve Resolution Of Ph Gradient Flow Injection Mixture Analysis With Correction Of The Schlieren Effect.

Multivariate curve resolution using alternating least squares (MCR-ALS) was used to quantify ascorbic (AA) and acetylsalicylic (ASA) acids in four pharmaceutical samples using a flow injection analysis (FIA) system with pH gradient and a diode array (DAD) spectrometer as a detector. Four different pharmaceutical drugs were analyzed, giving a data array of dimensions 51 x 291 x 61, corresponding respectively to number of samples, FIA times and spectral wavelengths. MCR-ALS was applied to these large data sets using different constraints to have optimal resolution and optimal quantitative estimations of the two analytes (AA and ASA). Since both analytes give an acid-basic pair of species contributing to the UV recorded signal, at least four components sholuld be proposed to model AA and ASA in synthetic mixture samples. Moreover, one additional component was needed to resolve accurately the Schlieren effect and another additional component was also needed to model the presence of possible interferences (like caffeine) in the commercial drugs tablets, giving therefore a total number of 6 independent components needed. The best quantification relative errors were around 2% compared to the reference values obtained by HPLC and by the oxidation-reduction titrimetric method, for ASA and AA respectively. In this work, the application of MCR-ALS allowed for the first time the full resolution of the FIA diffusion profile due to the Schlieren effect as an independent signal contribution, suggesting that the proposed MCR-ALS method allows for its accurate correction in FIA-DAD systems.133774-8

Understanding temporal and spatial changes of O3 or NO2 concentrations combining multivariate data analysis methods and air quality transport models

The application of the multivariate curve resolution method to the analysis of temporal and spatial data variability of hourly measured O3 and NO2 concentrations at nineteen air quality monitoring stations across Catalonia, Spain, during 2015 is shown. Data analyzed included ground-based experimental measurements and predicted concentrations by the CALIOPE air quality modelling system at three horizontal resolutions (Europe at 12 × 12 km2, Iberian Peninsula at 4 × 4 km2 and Catalonia at 1 × 1 km2). Results obtained in the analysis of these different data sets allowed a better understanding of O3 and NO2 concentration changes as a sum of a small number of different contributions related to daily sunlight radiation, seasonal dynamics, traffic emission patterns, and local station environments (urban, suburban and rural). The evaluation of O3 and NO2 concentrations predicted by the CALIOPE system revealed some differences among data sets at different spatial resolutions. NO2 predictions, showed in general a better performance than O3 predictions for the three model resolutions, specially at urban stations. Our results confirmed that the application of the trilinearity constraint during the multivariate curve resolution factor analysis decomposition of the analyzed data sets is a useful tool to facilitate the understanding of the resolved variability sources

Solution equilibria of the i-motif-forming region upstream of the B-cell lymphoma-2 P1 promoter

11 pages, 8 figures.-- PMID: 17850948 [PubMed].-- Printed version published Dec 2007.The 5'-end of the P1 promoter of the B-cell lymphoma-2 (bcl-2) gene contains a highly guaninecytosine-rich region, which has a role in the regulation of bcl-2 transcription. Whereas the guanine-rich region has been the focus of recent studies, little attention has been paid to the cytosine-rich strand. Here we examine the structural transitions of the cytosine-rich sequence by means of acidebase, mole-ratio and melting experiments monitored by molecular absorption, circular dichroism, and NMR spectroscopies. Two intramolecular i-motif structures have been detected in the pH range 2-7, with maximal formation at pH 4 and 6, respectively. At pH 7.6 the majority species has been associated with a hairpin involving Watson-Crick base pairs. Upon addition of the quadruplex-interacting ligand TmPyP4, bcl-2c structures at pH 6.1 and 7.6 yield identical interaction species with stoichiometries 1:2 (DNA:ligand) and logarithms of formation constant 12.4 ± 0.2 and 11.7 ± 0.1, respectively. The initial i-motif structure at pH 6.1 is lost upon interaction with TmPyP4.We acknowledge two grants from the Spanish Ministerio de Educación y Ciencia (projects BFU2004-02048/BMC and CTQ2006-15052-C02-01/BQU). We also thank Veronica Hernando for her contribution in carrying out some experiments.Peer reviewe