Browsing by Issue Date, starting with "2019-07-08"
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- Obstructive Sleep Apnea: a proteomics study of the effects of positive airway pressure therapyPublication . Valentim-Coelho, Cristina; Vaz, Fátima; Martins, Inês L.; Feliciano, Amélia; Pinto, Paula; Cristina, Bárbara; Penque, DeborahObstructive Sleep Apnea (OSA) syndrome is a common public health concern characterized by recurrent episodes of apneas and hypopneas during sleep. These obstructive events result in recurrent intermittent hypoxia and sleep fragmentation that can lead to metabolic and cardiovascular diseases. We recently demonstrated that OSA can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes1,2. Here we intend to investigate whether the first-line therapy for OSA, the positive airway pressure (PAP) can revert or modulate these proteome alterations. RBCs from Snorers and patients with severe OSA before/after 6 months of PAP treatment (n=10/condition) were depleted of hemoglobin, analyzed by 2D-DIGE using Progenesis SameSpotsv4.5. The differentially abundant proteins were identified by MALDI-MS/MS and protein annotations acquired by DAVIDv6.8. Western blotting (WB) validation was performed for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (overoxidized) GAPDHSO3 on a new Cohort (n=59). SPSS software was used to correlation studies with peroxiredoxin-2 (PRDX2) redox-oligomeric forms and several clinical parameters. Ten protein spots showed significant differences (Anova p<0.05) among groups and were associated with cell death, protein oligomerization and response to stress. Three proteoforms of GAPDH were identified decreased in OSA RBC (Anova p<0.05) and 6 months of PAP treatment increased these GAPDH proteoforms to the control levels. By WB, we confirmed these data by showing that the decreased GAPDH monomeric/tetrameric forms in OSA were increased by PAP treatment. PAP also increased GAPDHSO3 tetramers. In OSA, GAPDH monomers and GAPDHSO3 tetramers correlated positively with the respiratory disturbance index or triglycerides and adrenalin, respectively. After PAP, GAPDHSO3 tetramers correlated positively with PAP-induced PRDX2SO2/3 decameric forms, described having chaperone activity in cell protection. OSA induces alterations in the redox/oligomeric state of GAPDH and PRDX2 that can be reverted/modulated by PAP therapy. The clinical significant of these findings needs further validation and investigation.
- Nucleoplasmic bridges in alveolar cells induced by nanomaterial exposurePublication . Ventura, Célia; Matos, Paulo; Pereira, Joana F.S.; Marques, Bárbara; Sousa-Uva, António; Silva, Maria JoãoIntroduction: Multi-walled carbon nanotubes (MWCNT) are widely used engineered nanomaterials in industrial and biomedical applications. Yet, MWCNT inhalation, particularly in the occupational settings, may induce pulmonary adverse effects as acute or persistent pulmonary inflammation, interstitial fibrosis, granuloma, and bronchiolar or bronchioloalveolar hyperplasia, and the MWCNT-7 (Mitsui-7) has been classified as possibly carcinogenic to humans. In this work, we analyse the in vitro genotoxicity of MWCNT-7 using the cytokinesis-block micronucleus assay to evaluates the ability of MWCNT-7 to affect the viability of alveolar epithelial cells (necrosis, apoptosis), its mitotic status (mononucleated, binucleated, multinucleated) and its chromosomal damage or instability status (presence of micronuclei, nucleoplasmic bridges and nuclear buds).
- Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic studyPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Feliciano, Amelia; Antunes, Marília; Pinto, Paula; Barbara, Cristina; Penque, DeborahBackground: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.
- Occupational secondhand smoke exposure may modify the proteoma expression of human nasal epitheliumPublication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; Torres, Vukosava Milic; James, Peter; Simões, Tânia; Penque, DeborahThe tobacco is one of the biggest public health threats, smoking kills more than 7 million people/year worldwide and more than 890,000 are deaths resulting from exposure to Second Hand Smoke (SHS). In adults, SHS is associated to cardiovascular and respiratory diseases, including coronary heart disease and lung cancer, through pathological and molecular mechanisms not yet understood. We aimed to investigate the SHS effects on airway proteome in exposed workers. Nasal epithelium was collected from hospitality workers (non-smokers=40; smokers=12), long-term exposed and non-exposed to SHS at the workplace. Samples were analyzed by shotgun proteomics using an ESI-LQT Orbitrap XL mass spectrometer. The generated MS raw data was submitted to ‘PatternLab for Proteomics’ for peptide identification and relative quantification by label-free - extracted ion chromatograms (XIC). Golden rules were applied to obtain reliable data: in the identification of a protein at least one unique peptide must had to be present in more than 80% of the individuals, and consequently each inferred protein had to be detected in 80% to 100% of the cohort. Two proteins were found to be differentially expressed in the no-smokers exposed to SHS compared with the control: BPI fold-containing family A member 1 (BPIFA1) and Heat shock Protein Beta-1 (HSPB1). The first protein plays a role in the airway inflammatory response after exposure to irritants substances and the second is associated as a regulator of actin filament dynamics. Our findings support the indication that in non-smokers the prolonged exposure to SHS can lead to airway proteome modulation. When validated, the uncovered proteins can be promising candidates to “susceptibility/risk” and/or “predictive” biomarkers for SHS exposure.
- Sickle-cell disease investigated by computational proteomics approachesPublication . Costa, André; Neves, Sofia; Vaz, Fátima; Martins, Inês L.; James, Peter; Lavinha, João; Penque, DeborahSickle-cell disease (SCD) is a clinically heterogeneous autosomal recessive monogenic disorder characterized by recurrent episodes of severe haemolysis, vaso-occlusion and infection. Proteomics promises to offer novel unbiased molecular insights into the pathophysiology of SCD. The objective of this project is to analyze by bioinformatic tools mass spectrometry (MS) proteomics raw data, which has been generated by INSA’s Proteomic Laboratory in order to investigate proteome changes that might be related with SCD vaso-occlusion exacerbation. The MS raw data included in this study was generated by shotgun proteomic analysis on red blood cell (RBC) samples from six child SCD patients at steady-state and vaso-occlusion exacerbation episode and five child control subjects. The RBC samples, the haemoglobin depleted-cytoplasmic fraction and membrane fraction, were analysed by proteomic discovery-based approach using the ESI-LQT Orbitrap XL (Thermo) mass spectrometer. The generated MS raw data files were analysed by the PatternLab for Proteomic 4.0 bioinformatic platform for protein identification and extracted-ion chromatograms (XICs)-based label-free relative quantification. The following “golden parameters” were applied to obtain reliable and trustworthy data: one unique peptide at least should be considered to infer a protein identification, and the identified proteins should be present at least in 80% of the studied groups/conditions. 250 proteins were identified, and the respective normalized ion abundance factor was compared by using the Wilcoxon-T non-parametric statistical test. The differentially expressed proteins in crisis as compared to steady-state (p-value ≤ 5%) were investigated in the light of the Gene Ontology (GO) knowledge base (Database for Annotation, Visualization and Integrated Discovery – DAVID) and Reactome database for protein integration into signaling pathways with biological meaning. The most relevant results indicated that 3 cytoplasmic proteins (Dematin, Moesin and Protein S100-A4) and 9 membrane proteins (Eosinophil cationic protein, Bone marrow proteoglycan, Voltage-dependent anion-selective channel protein 1, Voltage-dependent anion-selective channel protein 2, Reticulon-3, Carbonic anhydrase 2, Haemoglobin subunit alpha, Haemoglobin subunit delta and Eosinophil peroxidase) showed significantly differential expression in crisis as compared to steady-state. Haemoglobin subunit alpha and Carbonic anhydrase 2 (CA2) have been reported as involved in important pathways related with O2/CO2 exchange in erythrocytes. Sickle cell crisis is frequently related to infection, involving Reticulon-3 and Haemoglobin subunit alpha. These proteins were identified to be modulated in the RBC membrane fraction from SCD patients at crisis. The most relevant proteins identified by these computational approaches will be selected for further biochemical verification by using SWATH-MS and/or Western blot methods. These proteins may be promising candidate early biomarkers to identify SCD patients at risk for vaso-occlusion crisis.