Publications by authors named "Alicia Robles"

7 Publications

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Organic Acids and Polyphenols Determination in Polish Wines by Ultrasound-Assisted Solvent Extraction of Porous Membrane-Packed Liquid Samples.

Molecules 2019 Nov 29;24(23). Epub 2019 Nov 29.

Department of Analytical Chemistry, Faculty of Chemistry, Gdańsk University of Technology, 11/12 Narutowicza Street, 80-233 Gdańsk, Poland.

In the near future, Poland is going to have more and more favorable conditions for viticulture. Organic acids and polyphenols are among the most commonly analyzed compounds due to their beneficial properties for human health and their importance in the winemaking process. In this work, a new technique involving ultrasound-assisted solvent extraction of porous membrane-packed liquid samples (UASE-PMLS) was for the first time described and applied for real samples. The methodology based on UASE-PMLS for organic acids and polyphenols in wine samples was optimized and validated. Using the new technique coupled to GC-MS, organic acids and polyphenols were evaluated in Polish wine samples. Extraction solvent, extraction temperature, derivatization time and sample pH were optimized. Chemometric tools were used for data treatment. Good linearity was obtained for the concentration ranges evaluated with values between 0.9852 and 0.9993. All parameters of method validation (intra- and inter-day precision and matrix effect) were over 80% with coefficient of variation (CV) up to 17%. Recovery was between (92.0 ± 8.5)% and (113 ± 16)%. Finally, green assessment was evaluated using Analytical Eco-Scale and Green Analytical Procedure Index (GAPI). The UASE-PMLS is characterized by many advantages, e.g., the extraction process is fast and easy coupled to GC-MS. Regarding other extraction techniques, the amount of used solvent is minimum, and no waste is generated. Therefore, it is an environmentally friendly technique.
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http://dx.doi.org/10.3390/molecules24234376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930624PMC
November 2019

Ruxolitinib in combination with prednisone and nilotinib exhibit synergistic effects in human cells lines and primary cells from myeloproliferative neoplasms.

Haematologica 2019 05 13;104(5):937-946. Epub 2018 Dec 13.

Hematology Service, Hospital Universitario 12 de Octubre.

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF.
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http://dx.doi.org/10.3324/haematol.2018.201038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518898PMC
May 2019

A novel high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia.

Oncotarget 2018 May 25;9(40):26019-26031. Epub 2018 May 25.

Strategic Research Program on CLL and B Cell Neoplasia Unit, Università Vita-Salute San Raffaele and IRCCS Istituto Scientifico San Raffaele, Milan, Italy.

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an model with the aim of reproducing the effects of the microenvironment that would help shed light on the mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses . Altogether, this novel assay represents an appropriate drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.
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http://dx.doi.org/10.18632/oncotarget.25419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995261PMC
May 2018

Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry.

SLAS Technol 2017 06 24;22(3):325-337. Epub 2017 Mar 24.

1 Vivia Biotech, Tres Cantos, Madrid, Spain.

Functional ex vivo assays that predict a patient's clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients' response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient's sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.
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http://dx.doi.org/10.1177/2472630317700346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464399PMC
June 2017

Lethal and sublethal effects of cadmium in the white shrimp Palaemonetes argentinus: a comparison between populations from contaminated and reference sites.

Ecotoxicol Environ Saf 2013 Mar 20;89:52-8. Epub 2012 Dec 20.

Laboratorio de Ecotoxicología, Departamento de Ciencias Marinas, Instituto de Investigaciones Marinas y Costeras, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350, Mar del Plata 7600, Argentina.

In the present study, the acute toxicity of cadmium (Cd) in white shrimp (Palaemonetes argentinus) from a metal polluted lagoon (Los Padres, LP) and from unpolluted lagoon (Nahuel Ruca, NR) was evaluated. Both population, were exposed to 3.06, 12.26, 30.66, 61.32, 306 and 613.2 μg Cd · L(-1) for 96-h. The sublethal effects of Cd were examined by two cellular biomarkers: metallothionein (MT) and lipid peroxidation (LPO). The seasonal variations of biomarkers in both lagoons were also evaluated. P. argentinus demonstrated a high sensitivity to Cd, with values of 96-h LC50 lower and close to those of highly sensitive species; therefore, can be proposed as a good indicator species. The LC(50) values of shrimp from LP (24-h: 269.8, 48-h: 67.45, 72-h: 30.66, 96-h: 24.50 μg Cd · L(-1)) were higher than those from NR (24-h: 153.3, 48-h: 32.65, 72-h: 18.40, 96-h: 12.26 μg Cd · L(-1)), indicating a higher tolerance to Cd, and it was related to their origin. Differential responses in terms of MT induction and LPO between populations were also detected. In NR shrimps, the MT synthesis was induced very fast (24-h) and even at the minimum concentration tested (3.06 μg Cd · L(-1)), while no increases were observed in LPO levels. In contrast, the MT and LPO levels in LP shrimps were not increased relative to control, although they were more tolerant to Cd than those of NR; suggesting the presence of another mechanism involved in the detoxification of Cd. The differences in both sensitivity and biochemical responses to Cd may be related with their environmental histories.
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http://dx.doi.org/10.1016/j.ecoenv.2012.11.008DOI Listing
March 2013

Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function.

BMC Microbiol 2006 Aug 31;6:75. Epub 2006 Aug 31.

Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid, Spain.

Background: Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.

Results: Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.

Conclusion: This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.
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http://dx.doi.org/10.1186/1471-2180-6-75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1564143PMC
August 2006

Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development.

J Cell Sci 2006 Mar 28;119(Pt 6):1154-64. Epub 2006 Feb 28.

Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid, Spain.

Genomic sequencing has revealed a large number of evolutionary conserved genes of unknown function. In the absence of characterized functional domains, the discovery of the role of these genes must rely on experimental approaches. We have selected 30 Dictyostelium discoideum genes of unknown function that showed high similarity to uncharacterized human genes and were absent in the complete proteomes from Saccharomyces cerevisiae and S. pombe. No putative functional motifs were found in their predicted encoded proteins. Eighteen genes were successfully knocked-out and three of them showed obvious phenotypes. A detailed analysis of one of them, midA, is presented in this report. Disruption of midA in Dictyostelium leads to pleiotropic defects. Cell size, growth rate, phagocytosis and macropinocytosis were affected in the mutant. During development, midA- cells showed an enhanced tendency to remain at the slug stage, and spore viability was compromised. The expression of MidA fused to GFP in midA- strain rescued the phenotype and the fused protein was located in the mitochondria. Although cellular oxygen consumption, mitochondrial content and mitochondrial membrane potential were similar to wild type, the amount of ATP was significantly reduced in the mutant suggesting a mitochondrial dysfunction. Metabolomic analysis by natural-abundance 13C-nuclear magnetic resonance has shown the lack of glycogen accumulation during growth. During starvation, mutant cells accumulated higher levels of ammonia, which inhibited normal development. We hypothesize that the lack of MidA reduces mitochondrial ATP synthetic capacity and this has an impact in some but not all energy-dependent cellular processes. This work exemplifies the potential of Dictyostelium as a model system for functional genomic studies.
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http://dx.doi.org/10.1242/jcs.02819DOI Listing
March 2006