Publications by authors named "Vitor H Teixeira"

37 Publications

Portuguese Football Federation consensus statement 2020: nutrition and performance in football.

BMJ Open Sport Exerc Med 2021 26;7(3):e001082. Epub 2021 Aug 26.

Portugal Football School, Portuguese Football Federation, Cruz Quebrada, Portugal.

Nutrition is an undeniable part of promoting health and performance among football (soccer) players. Nevertheless, nutritional strategies adopted in elite football can vary significantly depending on culture, habit and practical constraints and might not always be supported by scientific evidence. Therefore, a group of 28 Portuguese experts on sports nutrition, sports science and sports medicine sought to discuss current practices in the elite football landscape and review the existing evidence on nutritional strategies to be applied when supporting football players. Starting from understanding football's physical and physiological demands, five different moments were identified: preparing to play, match-day, recovery after matches, between matches and during injury or rehabilitation periods. When applicable, specificities of nutritional support to young athletes and female players were also addressed. The result is a set of practical recommendations that gathered consensus among involved experts, highlighting carbohydrates periodisation, hydration and conscious use of dietary supplements.
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http://dx.doi.org/10.1136/bmjsem-2021-001082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395276PMC
August 2021

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

Cancer Discov 2021 Oct 4;11(10):2456-2473. Epub 2021 May 4.

DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, United Kingdom.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when expression is induced during cancer development remains to be defined. Here we show that specific genes are upregulated in breast ductal carcinoma , and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487921PMC
October 2021

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Cancer Discov 2020 10 20;10(10):1489-1499. Epub 2020 Jul 20.

Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, signaling is upregulated, and the immunomodulator is downregulated. Changes appear intrinsic to the carcinoma lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer...
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http://dx.doi.org/10.1158/2159-8290.CD-19-1366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611527PMC
October 2020

Sestrins induce natural killer function in senescent-like CD8 T cells.

Nat Immunol 2020 06 30;21(6):684-694. Epub 2020 Mar 30.

Division of Infection and Immunity, University College London, London, UK.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8 T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27CD28CD8 T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27CD28CD8 T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27CD28CD8 T cells to acquire a broad-spectrum, innate-like killing activity.
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http://dx.doi.org/10.1038/s41590-020-0643-3DOI Listing
June 2020

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution.

Sci Adv 2019 12 11;5(12):eaaw3413. Epub 2019 Dec 11.

Department of Medicine, Boston University School of Medicine, Boston, MA, USA.

The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.
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http://dx.doi.org/10.1126/sciadv.aaw3413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905872PMC
December 2019

Behavioural and psychological pretreatment predictors of short- and long-term weight loss among women with overweight and obesity.

Eat Weight Disord 2020 Oct 13;25(5):1377-1385. Epub 2019 Sep 13.

Self-Regulation in Physical Activity, Nutrition and Obesity Research Group (PANO-SR), Interdisciplinary Center for the Study of Human Performance (CIPER), Faculdade de Motricidade Humana, Universidade de Lisboa, Estrada da Costa, Cruz Quebrada, 1495-687, Lisbon, Portugal.

Purpose: This study aims at identifying behavioural and psychological pretreatment predictors of 12- and 36-month weight loss in women with overweight/obesity enrolled in a behavioural weight management intervention.

Methods: A sample of 221 women participated in a randomized controlled trial on weight management (n = 184; n = 156). Multiple linear regressions were used to identify pretreatment predictors of successful weight loss, separately for intervention and control groups. Completers-only and baseline observation carried forward analyses were performed. This study is a secondary analysis of data from the 'Promotion of Exercise and Health in Obesity' randomized controlled trial.

Results: Fewer weight loss attempts in the last year positively predicted weight loss at 12 months in the intervention group, explaining 6% of the variance. At 36 months, in the intervention group, 20.2% of the variance in weight change was explained by lower eating disinhibition and higher weight-related quality of life in completers-only analyses, while baseline observation carried forward analyses explained only 9.8% of the variance in weight change via higher self-esteem and lower weight loss expectations. In the control group, higher exercise self-efficacy and a more internal weight locus of control predicted weight loss at 36 months, explaining 13.9% of the variance (completers-only analyses).

Conclusions: Previous weight loss attempts were identified as the most efficient pretreatment predictor of 12-month weight loss. Eating disinhibition, weight-related quality of life, self-esteem, weight loss expectations, exercise self-efficacy, and weight locus of control seem to be key factors for long-term success.

Level Of Evidence: Level I, randomized controlled trial.

Clinical Trial Registration: ClinicalTrials.gov identifier NCT00513084.
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http://dx.doi.org/10.1007/s40519-019-00775-9DOI Listing
October 2020

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

Nat Med 2019 03 21;25(3):517-525. Epub 2019 Jan 21.

The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.
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http://dx.doi.org/10.1038/s41591-018-0323-0DOI Listing
March 2019

The role of electrostatics in TrxR electron transfer mechanism: A computational approach.

Proteins 2016 12 25;84(12):1836-1843. Epub 2016 Oct 25.

Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016, Lisboa, Portugal.

Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a "shuttle," the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson-Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N-terminal (FAD and Cys59/64) and C-terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired "shuttle." Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the "shuttle" mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836-1843. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25166DOI Listing
December 2016

pK(a) Values of Titrable Amino Acids at the Water/Membrane Interface.

J Chem Theory Comput 2016 Mar 16;12(3):930-4. Epub 2016 Feb 16.

Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa , 1749-016 Lisboa, Portugal.

Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the pK(a) shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the pK(a) values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the pK(a) values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations.
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http://dx.doi.org/10.1021/acs.jctc.5b01114DOI Listing
March 2016

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

Am J Respir Crit Care Med 2016 07;194(2):156-68

1 Lungs for Living Research Centre, UCL Respiratory, University College London, London, United Kingdom.

Rationale: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.

Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts.

Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.

Measurements And Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model.

Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.
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http://dx.doi.org/10.1164/rccm.201507-1414OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003214PMC
July 2016

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

Free Radic Biol Med 2016 Jan 18;90:145-57. Epub 2015 Nov 18.

Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. Electronic address:

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.11.019DOI Listing
January 2016

Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers.

J Chem Theory Comput 2015 Dec 20;11(12):5973-9. Epub 2015 Nov 20.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa , Av. da República, 2780-157 Oeiras, Portugal.

Current constant-pH molecular dynamics (CpHMD) simulations provide a proper treatment of pH effects on the structure and dynamics of soluble biomolecules like peptides and proteins. However, addressing such effects on lipid membrane assemblies has remained problematic until now, despite the important role played by lipid ionization at physiological pH in a plethora of biological processes. Modeling (de)protonation events in these systems requires a proper consideration of the physicochemical features of the membrane environment, including a sound treatment of solution ions. Here, we apply our recent CpHMD-L method to the study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely reproducing the correct lipid phases of this system, namely, gel-fluid coexistence at pH 4 and a fluid phase at pH 7. A significant transition is observed for the membrane ionization and mechanical properties at physiological pH, providing a molecular basis for the well-established role of phosphatidic acid (PA) as a key player in the regulation of many cellular events. Also, as reported experimentally, we observed pH-induced PA-PA lipid aggregation at acidic pH. By including the titration of anionic phospholipids, the current methodology makes possible to simulate lipid bilayers with increased realism. To the best of our knowledge, this is the first simulation study dealing with a continuous phospholipid bilayer with pH titration of all constituent lipids.
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http://dx.doi.org/10.1021/acs.jctc.5b00956DOI Listing
December 2015

Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers.

J Chem Theory Comput 2014 Dec 13;10(12):5483-92. Epub 2014 Nov 13.

Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa , 1749-016 Lisboa, Portugal.

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems. These two problems have been circumvented by using ionized (deprotonated) anionic lipids and enough counterions to preserve the electroneutrality. In this work, we propose a method based on the Poisson-Boltzmann equation to estimate the counterion and co-ion concentration close to a lipid bilayer that avoids the need for neutrality at this microscopic level. The estimated number of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC lipid bilayer at different ionization levels. Our results show that the system neutralization represents an overestimation of the number of counterions. Consequently, the resulting lipid bilayer becomes too ordered and practically insensitive to ionization. On the other hand, our proposed approach is able to correctly model the ionization dependent isothermal phase transition of the bilayer observed experimentally. Furthermore, our approach is not too computationally expensive and can easily be used to model diverse charged biomolecular systems in molecular dynamics simulations.
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http://dx.doi.org/10.1021/ct500680qDOI Listing
December 2014

Constant-pH MD Simulations of an Oleic Acid Bilayer.

J Chem Theory Comput 2015 May 27;11(5):2367-76. Epub 2015 Apr 27.

Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa , Edifı́cio C8 Campo Grande, 1749-016 Lisboa, Portugal.

Oleic acid is a simple molecule with an aliphatic chain and a carboxylic group whose ionization and, consequently, intermolecular interactions are strongly dependent on the solution pH. The titration curve of these molecules was already obtained using different experimental methods, which have shown the lipid bilayer assemblies to be stable between pH 7.0 and 9.0. In this work, we take advantage of our recent implementations of periodic boundary conditions in Poisson-Boltzmann calculations and ionic strength treatment in simulations of charged lipid bilayers, and we studied the ionization dependent behavior of an oleic acid bilayer using a new extension of the stochastic titration constant-pH MD method. With this new approach, we obtained titration curves that are in good agreement with the experimental data. Also, we were able to estimate the slope of the titration curve from charge fluctuations, which is an important test of thermodynamic consistency for the sampling in a constant-pH MD method. The simulations were performed for ionizations up to 50%, because an experimentally observed macroscopic transition to micelles occurs above this value. As previously seen for a binary mixture of a zwitterionic and an anionic lipid, we were able to reproduce experimental results with simulation boxes usually far from neutrality. This observation further supports the idea that a charged membrane strongly influences the ion distribution in its vicinity and that neutrality is achieved significantly far from the bilayer surface. The good results obtained with this extension of the stochastic titration constant-pH MD method strongly supports its usefulness to sample the coupling between configuration and protonation in these types of biophysical systems. This method stands now as a powerful tool to study more realistic lipid bilayers where pH can influence both the lipids and the solutes interacting with them.
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http://dx.doi.org/10.1021/acs.jctc.5b00095DOI Listing
May 2015

Macrophage migration inhibitory factor-CXCR4 is the dominant chemotactic axis in human mesenchymal stem cell recruitment to tumors.

J Immunol 2015 Apr 23;194(7):3463-74. Epub 2015 Feb 23.

Lungs for Living Research Centre, Division of Medicine, University College London, London WC1E 6JF, United Kingdom;

Mesenchymal stromal cells (MSCs) are inherently tumor homing and can be isolated, expanded, and transduced, making them viable candidates for cell therapy. This tumor tropism has been used to deliver anticancer therapies to various tumor models. In this study, we sought to discover which molecules are the key effectors of human MSC tumor homing in vitro and using an in vivo murine model. In this study, we discover a novel role for macrophage migration inhibitory factor (MIF) as the key director of MSC migration and infiltration toward tumor cells. We have shown this major role for MIF using in vitro migration and invasion assays, in presence of different receptor inhibitors and achieving a drastic decrease in both processes using MIF inhibitor. Additionally, we demonstrate physical interaction between MIF and three receptors: CXCR2, CXCR4, and CD74. CXCR4 is the dominant receptor used by MIF in the homing tumor context, although some signaling is observed through CXCR2. We demonstrate downstream activation of the MAPK pathway necessary for tumor homing. Importantly, we show that knockdown of either CXCR4 or MIF abrogates MSC homing to tumors in an in vivo pulmonary metastasis model, confirming the in vitro two-dimensional and three-dimensional assays. This improved understanding of MSC tumor tropism will further enable development of novel cellular therapies for cancers.
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http://dx.doi.org/10.4049/jimmunol.1402097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374168PMC
April 2015

Molecular details of INH-C10 binding to wt KatG and Its S315T mutant.

Mol Pharm 2015 Mar 3;12(3):898-909. Epub 2015 Feb 3.

Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa , 1749-016 Lisboa, Portugal.

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of Mycobacterium tuberculosis to INH, mainly associated with mutations in the katG gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wt KatG or its S315T mutant form and either INH or INH-C10, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10 seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10 alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C10. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10 appears to be a very promising lead compound for drug development.
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http://dx.doi.org/10.1021/mp500736nDOI Listing
March 2015

Protonation of DMPC in a Bilayer Environment Using a Linear Response Approximation.

J Chem Theory Comput 2014 May;10(5):2176-84

Centro de Química e Bioquímica, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa , 1749-016 Lisboa, Portugal.

pH is a very important property, influencing all important biomolecules such as proteins, nucleic acids, and lipids. The effect of pH on proteins has been the subject of many computational works in recent years. However, the same has not been done for lipids, especially in their most biologically relevant environment: the bilayer. A reason for this is the inherent technical difficulty in dealing with this type of periodic systems. Here, we tackle this problem by developing a Poisson-Boltzmann-based method that takes in consideration the periodic boundary conditions of lipid bilayer patches. We used this approach with a linear response approximation to calculate the pKa value of a DMPC molecule when diluted in zwitterionic lipids. Our results show that DMPC protonation only becomes relevant at quite low pH values (2-3). However, when it happens, it has a strong impact on lipid conformations, leading to significant heterogeneity in the membrane.
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http://dx.doi.org/10.1021/ct5000082DOI Listing
May 2014

Processed pseudogenes acquired somatically during cancer development.

Nat Commun 2014 Apr 9;5:3644. Epub 2014 Apr 9.

Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5' truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.
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http://dx.doi.org/10.1038/ncomms4644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996531PMC
April 2014

Cell migration leads to spatially distinct but clonally related airway cancer precursors.

Thorax 2014 Jun 18;69(6):548-57. Epub 2014 Feb 18.

Lungs for Living Research Centre, UCL Respiratory, University College London, London, UK.

Background: Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5 years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related.

Methods: Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship.

Results: We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12 years.

Conclusions: Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.
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http://dx.doi.org/10.1136/thoraxjnl-2013-204198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033139PMC
June 2014

Dietary strategies to recover from exercise-induced muscle damage.

Int J Food Sci Nutr 2014 Mar 4;65(2):151-63. Epub 2013 Nov 4.

Faculdade de Desporto, Centro de Investigação, Formação, Intervenção e Inovação em Desporto (CIFI2D) .

Exhaustive or unaccustomed intense exercise can cause exercise-induced muscle damage (EIMD) and its undesirable consequences may decrease the ability to exercise and to adhere to a training programme. This review briefly summarises the muscle damage process, focusing predominantly on oxidative stress and inflammation as contributing factors, and describes how nutrition may be positively used to recover from EIMD. The combined intake of carbohydrates and proteins and the use of antioxidants and/or anti-inflammatory nutrients within physiological ranges are interventions that may assist the recovery process. Although the works studying food instead of nutritional supplements are very scarce, their results seem to indicate that food might be a favourable option as a recovery strategy. To date, the only tested foods were milk, cherries, blueberries and pomegranate with promising results. Other potential solutions are foods rich in protein, carbohydrates, antioxidants and/or anti-inflammatory nutrients.
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http://dx.doi.org/10.3109/09637486.2013.849662DOI Listing
March 2014

Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors.

Elife 2013 Oct 22;2:e00966. Epub 2013 Oct 22.

Lungs for Living Research Centre, UCL Respiratory , University College London , London , United Kingdom.

Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to "neutral drift" of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI:http://dx.doi.org/10.7554/eLife.00966.001.
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http://dx.doi.org/10.7554/eLife.00966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804062PMC
October 2013

Nutrition and nutritional issues for dancers.

Med Probl Perform Art 2013 Sep;28(3):119-23

Faculty of Nutrition and Food Sciences, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. Tel +351 225 074 320, fax +351 225 074 329. [email protected] fcna.up.pt.

Proper nutrition, not simply adequate energetic intake, is needed to achieve optimal dance performance. However, little scientific research exists concerning nutrition in dance, and so, to propose nutritional guidelines for this field, recommendations need to be based mainly on studies done in other physically active groups. To diminish the risk of energy imbalance and associated disorders, dancers must consume at least 30 kcal/kg fat-free mass/day, plus the training energy expenditure. For macronutrients, a daily intake of 3 to 5 g carbohydrates/kg, 1.2 to 1.7 g protein/kg, and 20 to 35% of energy intake from fat can be recommended. Dancers may be at increased risk of poor micronutrient status due to their restricted energy intake; micronutrients that deserve concern are iron, calcium, and vitamin D. During training, dancers should give special attention to fluid and carbohydrate intake in order to maintain optimal cognition, motivation, and motor skill performance. For competition/stage performance preparation, it is also important to ensure that an adequate dietary intake is being achieved. Nutritional supplements that may help in achieving specific nutritional goals when dietary intake is inadequate include multivitamins and mineral, iron, calcium, and vitamin D supplements, sports drinks, sports bars, and liquid meal supplements. Caffeine can also be used as an ergogenic aid. It is important that dancers seek dietary advice from qualified specialists, since the pressure to maintain a low body weight and low body fat levels is high, especially in styles as ballet, and this can lead to an unbalanced diet and health problems if not correctly supervised.
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September 2013

Conformational study of GSH and GSSG using constant-pH molecular dynamics simulations.

J Phys Chem B 2013 Jun 12;117(25):7507-17. Epub 2013 Jun 12.

Centro de Química e Bioquímica and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.

Glutathione is a small peptide with a crucial role in living organisms. This molecule is found in Nature in both reduced (GSH) and oxidized (GSSG) forms and a high GSH/GSSG ratio is essential to the cell. Glutathione is also present in several enzymatic reactions and can be found in many protein structures. As small peptides, these molecules do not have a defined structure in solution and are able to sample a broad conformational space. In addition, both molecules have several titration sites (four in GSH and six in GSSG) and their conformational space is inevitably influenced by pH. Here, we present a detailed conformational study of GSH and GSSG in a range of pH values, together with a full pH titration of these molecules. We performed constant-pH MD simulations of GSH and GSSG at 24 pH values in a total of 14.4 μs (300 ns per pH value). We obtained the two titration curves and the pKa values for all titrable groups with good agreement with experimental data. We also observed that GSH and GSSG have a large conformational variability in solution and their structural preferences are not significantly affected upon binding to proteins. Some exceptions were found and investigated in detail.
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http://dx.doi.org/10.1021/jp401066vDOI Listing
June 2013

LRIG1 regulates cadherin-dependent contact inhibition directing epithelial homeostasis and pre-invasive squamous cell carcinoma development.

J Pathol 2013 Mar;229(4):608-20

Lungs for Living Research Centre, UCL Respiratory, University College London, 5 University Street, London, WC1E 6JF, UK.

Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine-rich immunoglobulin repeats-1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re-expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E-cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition.
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http://dx.doi.org/10.1002/path.4148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806036PMC
March 2013

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach.

J Biol Inorg Chem 2012 Apr;17(4):543-55

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.

Hydrogenases are metalloenzymes that catalyze the reversible reaction H(2)<->2H(+) + 2e(-), being potentially useful in H(2) production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O(2) inhibition and produce more H(2) than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H(2) diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H(2) and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H(2) diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035-2045, 2006). The comparison showed that H(2) density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H(2) to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010-1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H(2) and proton pathways.
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http://dx.doi.org/10.1007/s00775-012-0875-2DOI Listing
April 2012

Analysis of binding modes of ligands to multiple conformations of CYP3A4.

Biochim Biophys Acta 2010 Oct 17;1804(10):2036-45. Epub 2010 Jun 17.

Centro de Biomedicina Molecular e Estrutural/Institute of Biotechnology and Bioengineering, Universidade do Algarve, Faculdade de Ciências e Tecnologia, Campus de Gambelas, 8005-139 Faro, Portugal.

Cytochromes P450 (CYPs) are extremely versatile enzymes capable of catalyzing a vast number of compounds, and CYP3A4 is no exception metabolizing approximately half of the currently marketed drugs, besides endogenous compounds. To metabolize such a variety of compounds, CYP3A4 has to be extremely flexible, which makes interaction studies difficult. We employ a multi-conformational docking setup where conformations are generated by several molecular dynamics simulations to analyze the binding modes of various ligands, and the docking is considered successful if the ligand site of catalysis (SOC) is within 6.0A of the haem Fe. While docking with the X-ray structure proved unsuccessful with all ligands, the multi-conformational docking achieved successful binding of each ligand to at least one protein conformation. Analysis of the docked solutions highlights residues in the active site cavity that may have an important role in access, binding and stabilization of the ligand.
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http://dx.doi.org/10.1016/j.bbapap.2010.06.008DOI Listing
October 2010

Antioxidants do not prevent postexercise peroxidation and may delay muscle recovery.

Med Sci Sports Exerc 2009 Sep;41(9):1752-60

Faculty of Nutrition and Food Sciences, University of Porto, Porto, Potugal.

Purpose: This study aimed to determine the effects of 4 wk of antioxidants (AOX) supplementation on exercise-induced lipid peroxidation, muscle damage, and inflammation in kayakers.

Methods: Subjects (n = 20) were randomly assigned to receive a placebo (PLA) or an AOX capsule (AOX; 272 mg of alpha-tocopherol, 400 mg of vitamin C, 30 mg of beta-carotene, 2 mg of lutein, 400 mug of selenium, 30 mg of zinc, and 600 mg of magnesium). Blood samples were collected at rest and 15 min after a 1000-m kayak race, both before and after the supplementation period, for analysis of alpha-tocopherol, alpha-carotene, beta-carotene, lycopene, lutein plus zeaxanthin, vitamin C, uric acid, total AOX status (TAS), thiobarbituric reactive acid substances (TBARS) and interleukin-6 (IL-6) levels, and creatine kinase (CK), superoxide dismutase (SOD), glutathione reductase (Gr), and glutathione peroxidase (GPx) activities.

Results: With supplementation, plasma alpha-tocopherol (P = 0.003) and beta-carotene (P = 0.007) augmented significantly in the AOX group. IL-6 (exercise, P = 0.039), TBARS (exercise, P < 0.001), and uric acid (exercise, P = 0.032) increased significantly in response to the exercise regardless of treatment group. Cortisol level raised more from pre- to postsupplementation period in the PLA group (time x supplementation, P = 0.002). Although TAS declined after exercise before intervention, it increased above preexercise values after the 4-wk period in the AOX group (supplementation x time x exercise, P = 0.034). CK increased after exercise in both groups (exercise effect, P < 0.001) and decreased from week 0 to week 4 more markedly in the PLA group (supplementation x time, P = 0.049).

Conclusions: AOX supplementation does not offer protection against exercise-induced lipid peroxidation and inflammation and may hinder the recovery of muscle damage.
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http://dx.doi.org/10.1249/MSS.0b013e31819fe8e3DOI Listing
September 2009

Proton pathways in a [NiFe]-hydrogenase: A theoretical study.

Proteins 2008 Feb;70(3):1010-22

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, E.A.N, Ap. 127, 2780-157 Oeiras, Portugal.

We present here a theoretical study to investigate possible proton pathways in the [NiFe]-hydrogenase from Desulfovibrio gigas. The approach used in this study consists of a combination of Poisson-Boltzmann and Monte Carlo simulations together with a distance-based network analysis to find possible groups involved in the proton transfer. Results obtained at different pH values show a reasonable number of proton active residues distributed by the protein interior and surface, with a concentration around the metal centres. The electrostatic interactions in this protein are strong, as shown by the unusual shape of the titration curves of several sites. Some residue pairs show strongly correlated protonations, indicating the sharing and probably exchange of a proton between them. The conjugation of the PB and MC simulations with the distance-based analysis allows a detailed characterization of the possible proton pathways. We discuss previous suggestions and propose a new complete pathway for the proton transfer between the active site and the surface. This pathway is mainly composed of histidines and glutamic acid residues.
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http://dx.doi.org/10.1002/prot.21588DOI Listing
February 2008

Natural domain design: enhanced thermal stability of a zinc-lacking ferredoxin isoform shows that a hydrophobic core efficiently replaces the structural metal site.

Biochemistry 2006 Aug;45(34):10376-84

Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.

Zinc centers play a key role as important structure determinants in a variety of proteins including ferredoxins (Fd). Here, we exploit the availability of two highly similar ferredoxin isoforms from the thermophile Sulfolobus metallicus, which differ in the residues involved in coordinating a His/Asp zinc site that ties together the protein core with its N-terminal extension, to investigate the effect of the absence of this site on ferredoxin folding. The conformational properties of the zinc-containing (FdA) and zinc-lacking (FdB) isoforms were investigated using visible absorption and tryptophan fluorescence emission. Fluorescence quenching studies, together with comparative modeling and molecular dynamics simulations, indicate that the FdB N-terminal extension assumes a fold identical to that of the Zn(2+)-containing isoform. The thermal stability of the isoforms was investigated in a broad pH range (2 < pH < 10), and at physiological pH conditions, both proteins unfold above 100 degrees C. Surprisingly, the Zn(2+)-lacking isoform was always found to be more stable than its Zn(2+)-containing counterpart: a DeltaT(m) approximately 9 degrees C is determined at pH 7, a difference that becomes even more significant at extreme pH values, reaching a DeltaT(m) approximately 24 degrees C at pH 2 and 10. The contribution of the Zn(2+) site to ferredoxin stability was further resolved using selective metal chelators. During thermal unfolding, the zinc scavenger TPEN significantly lowers the T(m) in FdA ( approximately 10 degrees C), whereas it has no effect in FdB. This shows that the Zn(2+) site contributes to ferredoxin stability but that FdB has devised a structural strategy that accounts for an enhanced stability without using a metal cross-linker. An analysis of the FdB sequence and structural model leads us to propose that the higher stability of the zinc-containing ferredoxin results from van der Waals contacts formed between the residues that occupy the same spatial region where the zinc ligands are found in FdA. These favor the formation of a novel local stabilizing hydrophobic core and illustrate a strategy of natural fold design.
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http://dx.doi.org/10.1021/bi0610698DOI Listing
August 2006

On the use of different dielectric constants for computing individual and pairwise terms in poisson-boltzmann studies of protein ionization equilibrium.

J Phys Chem B 2005 Aug;109(30):14691-706

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, Apartado 127, 2781-901 Oeiras, Portugal.

Poisson-Boltzmann (PB) models are a fast and common tool for studying electrostatic processes in proteins, particularly their ionization equilibrium (protonation and/or reduction), often yielding quite good results when compared with more detailed models. Yet, they are conceptually very simple and necessarily approximate, their empirical character being most evident when it comes to the choice of the dielectric constant assigned to the protein region. The present study analyzes several factors affecting the ability of PB-based methods to model protein ionization equilibrium. We give particular attention to a suggestion made by Warshel and co-workers (e.g., Sham et al. J. Phys. Chem. B 1997, 101, 4458) of using different protein dielectric constants for computing the individual (site) and the pairwise (site-site) terms of the ionization free energies. Our prediction of pK(a) values for several proteins indicates that no advantage is obtained by such a procedure, even for sites that are buried and/or display large pK(a) shifts relative to the solution values. In particular, the present methodology gives the best predictions using a dielectric constant around 20, for shifted/buried and nonshifted/exposed sites alike. The similarities and differences between the PB model and Warshel's PDLD/S model are discussed, as well as the reasons behind their apparently discrepant results. The present PB model is shown to predict also good reduction potentials in redox proteins.
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http://dx.doi.org/10.1021/jp052259fDOI Listing
August 2005
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