Publications

Comparison of two metabolomics platforms to discover biomarkers in critically ill patients from serum analysis

Highlights
- UHPLC-HRMS identified 13 metabolites predicting IMV and 8 associated to mortality.
- FTIR data permitted classification of patients by IMV and death outcomes.
- FTIR data excelled UHPLC-HRMS in predictive models with unbalanced groups.
-Blood serum metabolomics as a promising tool for predicting ICU patients' prognosis.

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Fonseca TAF, Von Rekowski CP, Araújo R, Oliveira MC, Justino GC, Bento L, Calado CRC. Comparison of two metabolomics platforms to discover biomarkers in critically ill patients from serum analysis. Comp in Biol and Med 2025, 184, 109393.

The Elusive Biological Activity of Scorpionates: A Useful Scaffold for Cancer Therapy?

Cancer remains a formidable challenge, requiring the constant pursuit of novel therapeutic agents and strategies. Scorpionates, known for their unique coordination properties, have recently gained attention for their anticancer potential. Traditionally applied in catalysis, these compounds have demonstrated notable cytotoxicity across various cancer cell lines, often surpassing the efficacy of conventional chemotherapeutics. This review addresses recent findings on scorpionate complexes, emphasizing the impact of metal choice and ligand design on biological activity. Copper and ruthenium scorpionates show promise, leveraging redox activity and mitochondrial disruption mechanisms to selectively induce cancer cell death. Ligand modifications, including sulfur-containing heterocycles and unsubstituted pyrazoles, have proven effective in enhancing cytotoxicity and selectivity. Furthermore, dipodal ligands show unique potential, with selective binding sites that improve stability and facilitate specific cellular interactions, such as targeting metastatic pathways. These findings highlight the largely unexplored potential of scorpionate complexes, positioning them as candidates for next-generation anticancer therapies. Continued research into structure–activity relationships and precise mechanisms of action could pave the way for developing highly potent and selective anticancer agents based on scorpionate chemistry.

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Petrosian A, Pinheiro PF, Ribeiro APC, Martins LMDRS, Justino GC. The Elusive Biological Activity of Scorpionates: A Useful Scaffold for Cancer Therapy? Molecules, 2024, 29(23), 5672.

Sun Y, Madureira J, Justino GC, Cabo-Verde S, Chmielewska-Śmietanko D, Sudlitz M, Bulka S, Chajduk E, Mróz A, Wang S, Wang J. Diclofenac Degradation in Aqueous Solution Using Electron Beam Irradiation and Combined with Nanobubbling.
Appl Sci 2024, 14, 6028.

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Exploring the Mechanisms behind the Anti-Tumoral Effects of Model C-Scorpionate Complexes

The growing worldwide cancer incidence, coupled to the increasing occurrence of multidrug cancer resistance, requires a continuous effort towards the identification of new leads for cancer management. In this work, two C-scorpionate complexes were studied as potential scaffolds for future anticancer drug development. Their cytotoxicity and cell migration inhibitory activity were analyzed, and an untargeted metabolomics approach was employed to elucidate the biological processes significantly affected by these two complexes, using two tumoral cell lines (B16 and HCT116) and a non-tumoral cell line (HaCaT). While the Fe complex did not display a significant cytotoxicity, the Co complex was particularly cytotoxic against the HCT116 cell line. While the Co complex significantly inhibited cell migration in all tested cell lines, the Fe complex displayed a mixed activity. From a metabolomics perspective, exposure to [the Fe complex was associated with changes in various metabolic pathways involving tyrosine, where iron-dependent enzymes are particularly relevant. On the other hand, the Co complex was associated with dysregulation of cell adhesion and membrane structural pathways, suggesting that its antiproliferative and anti-migration properties could be due to changes in the overall cellular adhesion mechanisms.

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Silva PMG, Pinheiro PF, Camões SP, Ribeiro APC, Martins LMDRS, Miranda JPG, Justino GC*. Exploring the Mechanisms behind the Anti-Tumoral Effects of Model C-Scorpionate Complexes. Molecules 2023, 28, 5451.

An Optimised MS-Based Versatile Untargeted Metabolomics Protocol

Untargeted metabolomics approaches require complex samples containing the endogenous metabolites of a biological system. Here, we describe a set of protocols that can be applied to various types of samples, including prokaryotic and eukaryotic cells, as well as animal and human samples. Following a single extraction step, samples are analysed using different chromatographic conditions coupled to high-resolution mass spectrometry. Quantification of metabolite changes between samples is performed without internal standards, using peak areas from extracted ion chromatograms for statistical analysis. Bioinformatics annotation of the results allows a pathway- and process-oriented analysis across biological sample conditions, allowing a complete pathway interrogation.

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Marques CF, Justino GC. An Optimised MS-Based Versatile Untargeted Metabolomics Protocol. Separations 2023, 10, 314.

The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLC-MS/MS and FTIR Spectroscopy

Biofluid metabolomics is a very appealing tool to increase the knowledge associated with pathophysiological mechanisms leading to better and new therapies and biomarkers for disease diagnosis and prognosis. However, due to the complex process of metabolome analysis, including the metabolome isolation method and the platform used to analyze it, there are diverse factors that affect metabolomics output. In the present work, the impact of two protocols to extract the serum metabolome, one using methanol and another using a mixture of methanol, acetonitrile, and water, was evaluated. The metabolome was analyzed by ultraperformance liquid chromatography associated with tandem mass spectrometry (UPLC-MS/MS), based on reverse-phase and hydrophobic chromatographic separations, and Fourier transform infrared (FTIR) spectroscopy. The two extraction protocols of the metabolome were compared over the analytical platforms (UPLC-MS/MS and FTIR spectroscopy) concerning the number of features, the type of features, common features, and the reproducibility of extraction replicas and analytical replicas. The ability of the extraction protocols to predict the survivability of critically ill patients hospitalized at an intensive care unit was also evaluated. The FTIR spectroscopy platform was compared to the UPLC-MS/MS platform and, despite not identifying metabolites and consequently not contributing as much as UPLC-MS/MS in terms of information concerning metabolic information, it enabled the comparison of the two extraction protocols as well as the development of very good predictive models of patient’s survivability, such as the UPLC-MS/MS platform. Furthermore, FTIR spectroscopy is based on much simpler procedures and is rapid, economic, and applicable in the high-throughput mode, i.e., enabling the simultaneous analysis of hundreds of samples in the microliter range in a couple of hours. Therefore, FTIR spectroscopy represents a very interesting complementary technique not only to optimize processes as the metabolome isolation but also for obtaining biomarkers such as those for disease prognosis.

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Fonseca TAH, Von Rekowski CP, Araújo R, Oliveira MC, Justino GC, Bento L, Calado CRC. The Impact of the Serum Extraction Protocol on Metabolomic Profiling Using UPLCMS/ MS and FTIR Spectroscopy. ACS Omega. 2023 8:20755-20766.

Protocol to study in vitro drug metabolism and identify montelukast metabolites from purified enzymes and primary cell cultures by mass spectrometry

Highlights
- An in vitro set of assays that can be used in drug metabolism studies
- A high-resolution electrospray ionization mass spectrometry analytical tool
- Characterization of drug metabolites by mass spectrometry
- Small-scale metabolite identification

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Marques CF, Pinheiro PF, Justino GC. Protocol to study in vitro drug metabolism and identify montelukast metabolites from purified enzymes and primary cell cultures by mass spectrometry. STAR Protoc. 2023 4(1):102086.

Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression

Resistance to treatment, particularly to radiotherapy, is still a major clinical problem in cancer management. Macrophages are abundant immune cells at the tumor microenvironment, being exposed to ionizing radiation during cancer radiotherapy. Considering the role of macrophages in tumor progression and therapy outcome, it is crucial to investigate their response to clinically relevant ionizing radiation doses for the design of new strategies to overcome tumor radio resistance. In this work, we have used a proteomic approach to evaluate the expression profile of irradiated versus non-irradiated macrophages. This analysis, supported by validation using cell-based assays, led to the identification of two main deregulated targets, cathepsin D and transferrin receptor 1, in irradiated macrophages. Investigating macrophage response to ionizing radiation could lead to the identification of deregulated pathways and molecular players that can be targeted to overcome tumor radio resistance.

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Pinto AT*, Machado AB, Osório H, Pinto ML, Vitorino R, Justino GC, Santa C, Castro F, Cruz T, Lima J, Cardoso AP, Figueira R, Monteiro A, Marques M, Manadas B, Pauwels J, Gevaert K, Mareel M, Rocha S, Duarte T, Oliveira, MJ. Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression. Cancers2023 15(1):270.

RN Bento, MA Rendas, VAR Semedo, CF Marques, GC Justino, CES Bernardes, MEM Piedade, F Antunes. Energy dissipation in early detection of cellular responses to metabolic challenges. bioRxiv2022

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The mechanisms underlying montelukast's neuropsychiatric effects – new insights from a combined metabolic and multiomics approach

Montelukast (MTK) is an antagonist of the cysteinyl leukotrienes receptor 1 widely used to manage asthma symptoms among adults and children. However, it has been associated with an increasing number of neuropsychiatric adverse drug reactions (ADRs), particularly among children, including depression, sleep disturbance, and suicidal ideation. The aims of this work were to characterize MTK metabolism in vitro and in vivo and to identify its effects at the metabolome and proteome levels in order to explain its toxicity. Results clearly indicate that montelukast therapeutic effects are accompanied by a strong modulation of specific processes in the central nervous system that may explain the observed neuropsychiatric reactions. Moreover, the results also suggest that adverse drug reactions are more likely to occur in children, due to the early maturation stage of their brains.

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Marques CF, Marques MM, Justino GC. The mechanisms underlying montelukast's neuropsychiatric effects – new insights from a combined metabolic and multiomics approach. Life Sci. 2022 310:121056.

Optimized protocol for obtaining and characterizing primary neuron-enriched cultures from embryonic chicken brains

Highlights
An in vitro model that can be used for Alzheimer disease studies
Fast and accessible protocol with no need for an animal facility
Isolation of a neuron-enriched cell fraction from chicken embryos
Characterization of the neuronal model using proteomics and cell staining approaches

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Marques CF, Pinheiro PF, Justino GC. Optimized protocol for obtaining and characterizing primary neuron-enriched cultures from embryonic chicken brains. STAR Protoc. 2022 3(4):101753.

Marques CF, Marques MM, Justino GC. Leukotrienes vs. Montelukast-Activity, Metabolism, and Toxicity Hints for Repurposing. Pharmaceuticals (Basel). 2022 15(9):1039.

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Molecular dynamics simulations and analysis for bioinformatics undergraduate students

A computational biochemistry laboratory, fitted for bioinformatics students, is presented. The molecular dynamics package GROMACS is used to prepare and simulate a solvated protein. Students analyze the trajectory with different available tools (GROMACS and VMD) to probe the structural stability of the protein during the simulation. Students are also required to make use of Python libraries and write their own code to probe non-covalent interactions between the amino acid side chains. Based on these results, students characterize the system in a qualitatively approach but also assess the importance of each specific interaction through time. This work mobilizes biochemical concepts and programming skills, fostering critical thinking and group work and developing presenting skills.

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Justino GC, Nascimento CP, Justino MC. Molecular dynamics simulations and analysis for bioinformatics undergraduate students. Biochem Mol Biol Educ.2021 49(4):570-582.

Immobilization of His-tagged proteins on NiO foams for recyclable enzymatic reactors

Highlights
NiO foams with high surface area were synthesised.
His-tag proteins bind to the NiO foam and remain bound upon wash.
Protein binding to the foam results in microporous cavities alterations.
DFT suggests His-tags prefer stretched conformations and act as bidentate ligands.
Immobilized His-tag enzyme remains active and the support is recyclable.

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Rosado PR, Meyrelles R, Macatrão AM, Justino MC, Gomes AG, Montemor MF, Alves MM, Justino GC, Ribeiro APC, Shimizu K. Immobilization of His-tagged proteins on NiO foams for recyclable enzymatic reactors Appl Surf Sci2021 537:147848.

Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization.

Cancer is the second cause of death worldwide. This devastating disease requires specific, fast, and affordable solutions to mitigate and reverse this trend. A step towards cancer-fighting lies in the isolation of natural killer (NK) cells, a set of innate immune cells, that can either be used as biomarkers of tumorigenesis or, after autologous transplantation, to fight aggressive metastatic cells. In order to specifically isolate NK cells (which express the surface NKp30 receptor) from peripheral blood mononuclear cells, a ZnO immunoaffinity-based platform was developed by electrodeposition of the metal oxide on a flexible indium tin oxide (ITO)-coated polyethylene terephthalate (PET) substrate. The resulting crystalline and well-aligned ZnO nanorods (NRs) proved their efficiency in immobilizing monoclonal anti-human NKp30 antibodies (mAb), obviating the need for additional procedures for mAb immobilization. The presence of NK cells on the peripheral blood mononuclear cell (PBMCs) fraction was evaluated by the response to their natural ligand (B7-H6) using an acridine orange (AO)-based assay. The successful selection of NK cells from PBMCs by our nanoplatform was assessed by the photoluminescent properties of AO. This easy and straightforward ZnO-mAb nanoplatform paves the way for the design of biosensors for clinic diagnosis, and, due to its inherent biocompatibility, for the initial selection of NK cells for autotransplantation immunotherapies.

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Babu KS, Pinheiro PF, Marques CF, Justino GC, Andrade SM, Alves MM. Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization. Sci Rep.2020 10(1):15018

NKp30 - A prospective target for new cancer immunotherapy strategies.

Natural killer (NK) cells are an important arm of the innate immune system. They constitutively express the NKp30 receptor. NKp30-mediated responses are triggered by the binding of specific ligands e.g. tumour cell-derived B7-H6 and involve the secretion of cytotoxic mediators including TNF-α, IFN-γ, perforins and granzymes. The latter two constitute a target cell-directed response that is critical in the process of immunosurveillance. The structure of NKp30 is presented, focusing on the ligand-binding site, on the ligand-induced structural changes and on the experimental data available correlating structure and binding affinity. The translation of NKp30 structural changes to disease progression is also reviewed. NKp30 role in immunotherapy has been explored in chimeric antigen receptor T-cell (CAR-T) therapy. However, antibodies or small ligands targeting NKp30 have not yet been developed. The data reviewed herein unveil the key structural aspects that must be considered for drug design in order to develop novel immunotherapy approaches.

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Pinheiro PF, Justino GC, Marques MM. NKp30 - A prospective target for new cancer immunotherapy strategies. Br J Pharmacol.2020 177(20):4563-4580.

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