Percorrer por autor "Calado, Cecília R. C."
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- Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugsPublication . Ladeira, Carina; Araújo, Rúben; Ramalhete, Luís; Teixeira, Hélder; Calado, Cecília R. C.Genotoxicity is important information that should be included in human biomonitoring programs. However, the usually applied cytogenetic assays are laborious and time-consuming, the reason why it is critical to developing rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p<0.01), and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p<0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from exposure with an accuracy, sensitivity, and specificity of 92 %, 93 %, and 91 %, respectively. All these parameters were achieved based on 1 μL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be concluded that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.
- Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs: a comparison with cytokinesis-block micronucleus assay resultsPublication . Ladeira, Carina; Araújo, Rúben; Ramalhete, Luís; Teixeira, Hélder; Calado, Cecília R. C.Genotoxicity is an important information that should be included in human biomonitoring programs design. Cytogenetic methods are usually laborious and time-consuming; therefore, the development of new molecular methods is an added value. This study aimed to evaluate if the molecular profile of previously frozen whole blood acquired by Fourier Transform Infrared (FTIR) spectroscopy allows for to assessment of genotoxicity in occupational exposure to antineoplastic drugs in hospital professionals, as obtained by the lymphocyte cytokinesis block micronucleus (CBMN) assay.
- Epigenetic and drug response modulation of Epigalocaten-in-3-gallate in Staphylococcus aureus with divergent resistance phenotypesPublication . Mira, Ana Rita; Zeferino, Ana Sofia; Inácio, Raquel; Delgadinho, Mariana; Brito, Miguel; Calado, Cecília R. C.; Ribeiro, EdnaHealthcare-associated methicillin-resistant Staphylococcus aureus infections represent extremely high morbidity and mortality rates worldwide. We aimed to assess the antimicrobial potential and synergistic effect between Epigalocatenin-3-gallate (EGCG) and different antibiotics in S. aureus strains with divergent resistance phenotypes. EGCG exposure effects in epigenetic and drug resistance key modulators were also evaluated. S. aureus strains (n = 32) were isolated from infected patients in a Lisbon hospital. The identification of the S. aureus resistance phenotype was performed through automatized methods. The antibiotic synergistic assay was performed through disk diffusion according to EUCAST guidelines with co-exposure to EGCG (250, 100, 50, and 25 µg/mL). The bacteria's molecular profile was assessed through FTIR spectroscopy. The transcriptional expression of OrfX, SpdC, and WalKR was performed by using qRT-PCR. FTIR-spectroscopy analysis enabled the clear discrimination of MRSA/MSSA strains and the EGCG exposure effect in the bacteria's molecular profiles. Divergent resistant phenotypes were associated with divergent transcriptional expression of the epigenetic modulator OrfX, particularly in MRSA strains, as well as the key drug response modulators SpdC and WalKR. These results clearly demonstrate that EGCG exposure alters the expression patterns of key epigenetic and drug response genes with associated divergent-resistant profiles, which supports its potential for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms.
- Impact of epigallocatechinn-3-gallate on Staphylococcus aureus molecular profilePublication . Inácio, Raquel; Ribeiro, Edna; Calado, Cecília R. C.The discovery of new antimicrobial compounds is critical for the control of severe nosocomial infections, as those associated with methicillin-sensitive (MSSA) and resistant (MRSA) Staphylococcus aureus strains. In order to enhance new therapeutic approaches, it is crucial to develop new platforms to screen innovative compounds. Here, we evaluated how Fourier Transform Infra-Red (FTIR)-spectroscopy enables the prediction of antibiotic resistance and monitors the impact of epigallocatechin-3-gallate (EGCG) on the metabolism of MRSA and MSSA strains. Data showed that EGCG impacts the bacteria's metabolism, and that MRSA strains are more sensitive to EGCG. The high sensitivity of the technique also enabled us to discriminate the impact of EGCG concentrations, i.e., between 25 and 50, and between 50 and 100μg/ml. On the other hand, EGCG's impact on cellular molecular composition was lower than the differences between MSSA and MRSA strains. Furthermore, it was possible to predict these strains' resistance towards the antibiotics methicillin, amoxicillin, imipenem, and gentamicin. Since the spectra were acquired in a rapid, simple, economic, and high-throughput mode, this methodology may strongly promote the surveillance of nosocomial infection caused by S. aureus, and to screen new antimicrobial compounds.
- Rapid FTIR spectral fingerprinting of kidney allograft perfusion fluids distinguishes DCD from DBD donors: a pilot machine learning studyPublication . Ramalhete, Luís ; Araújo, Rúben; Vieira, Miguel Bigotte ; Vigia, Emanuel ; Pena, Ana ; Carrelha, Sofia; Ferreira, Aníbal ; Calado, Cecília R. C.Background/Objectives: Rapid, objective phenotyping of donor kidneys is needed to support peri-implant decisions. Label-free Fourier-transform infrared (FTIR) spectroscopy of static cold-storage Celsior® perfusion fluid can discriminate kidneys recovered from donation after circulatory death (DCD) versus donation after brain death (DBD). Methods: Preservation solution from isolated kidney allografts (n = 10; 5 DCD/5 DBD) matched on demographics was analyzed in the Amide I and fingerprint regions. Several spectral preprocessing steps were applied, and feature extraction was based on the Fast Correlation-Based Filter. Support vector machines and Naïve Bayes were evaluated. Unsupervised structure was assessed based on cosine distance, multidimensional scaling, and hierarchical clustering. Two-dimensional correlation spectroscopy (2D-COS) was used to examine band co-variation. Results: Donor cohorts were well balanced, except for higher terminal serum creatinine in DCD. Quality metrics were comparable, indicating no systematic technical bias. In Amide I, derivatives improved classification, but performance remained modest (e.g., second derivative with feature selection yielded an area under the curve (AUC) of 0.88 and an accuracy of 0.90 for support vector machines; Naïve Bayes reached an AUC of 0.92 with an accuracy of 0.70). The fingerprint window was most informative. Naïve Bayes with second derivative plus feature selection identified bands at ~1202, ~1203, ~1342, and ~1413 cm−1 and achieved an AUC of 1.00 and an accuracy of 1.00. Unsupervised analyses showed coherent grouping in the fingerprint region, and 2D correlation maps indicated coordinated multi-band changes. Conclusions: Performance in this 10-sample pilot should be interpreted cautiously, as perfect leave-one-out cross-validation (LOOCV) estimates are vulnerable to overfitting. The findings are preliminary and hypothesis-generating, and they require confirmation in larger, multicenter cohorts with a pre-registered analysis pipeline and external validation.
- A simple, label-free, and high-throughput method to evaluate the epigallocatechin-3-gallate impact in plasma molecular profilePublication . Araújo, Rúben; Ramalhete, Luís; Paz, Helder da; Ribeiro, Edna; Calado, Cecília R. C.Epigallocatechin-3-gallate (EGCG), the major catechin present in green tea, presents diverse appealing biological activities, such as antioxidative, anti-inflammatory, antimicrobial, and antiviral activities, among others. The present work evaluated the impact in the molecular profile of human plasma from daily consumption of 225 mg of EGCG for 90 days. Plasma from peripheral blood was collected from 30 healthy human volunteers and analyzed by high-throughput Fourier transform infrared spectroscopy. To capture the biochemical information while minimizing the interference of physical phenomena, several combinations of spectra pre-processing methods were evaluated by principal component analysis. The pre-processing method that led to the best class separation, that is, between the plasma spectral data collected at the beginning and after the 90 days, was a combination of atmospheric correction with a second derivative spectra. A hierarchical cluster analysis of second derivative spectra also highlighted the fact that plasma acquired before EGCG consumption presented a distinct molecular profile after the 90 days of EGCG consumption. It was also possible by partial least squares regression discriminant analysis to correctly predict all unlabeled plasma samples (not used for model construction) at both timeframes. We observed that the similarity in composition among the plasma samples was higher in samples collected after EGCG consumption when compared with the samples taken prior to EGCG consumption. Diverse negative peaks of the normalized second derivative spectra, associated with lipid and protein regions, were significantly affected (p < 0.001) by EGCG consumption, according to the impact of EGCG consumption on the patients' blood, low density, and high-density lipoproteins ratio. In conclusion, a single bolus dose of 225 mg of EGCG, ingested throughout a period of 90 days, drastically affected plasma molecular composition in all participants, which raises awareness regarding prolonged human exposure to EGCG. Because the analysis was conducted in a high-throughput, label-free, and economic analysis, it could be applied to high-dimension molecular epidemiological studies to further promote the understanding of the effect of bio-compound consumption mode and frequency.