The potential of metabolomics in the diagnosis of thyroid cancer

Thyroid cancer is the most common endocrine system malignancy. However, there is still a lack of reliable and specific markers for the detection and staging of this disease. Fine needle aspiration biopsy is the current gold standard for diagnosis of thyroid cancer, but drawbacks to this technique include indeterminate results or an inability to discriminate different carcinomas, thereby requiring additional surgical procedures to obtain a final diagnosis. It is, therefore, necessary to seek more reliable markers to complement and improve current methods. “Omics” approaches have gained much attention in the last decade in the field of biomarker discovery for diagnostic and prognostic characterisation of various pathophysiological conditions. Metabolomics, in particular, has the potential to identify molecular markers of thyroid cancer and identify novel metabolic profiles of the disease, which can, in turn, help in the classification of pathological conditions and lead to a more personalised therapy, assisting in the diagnosis and in the prediction of cancer behaviour. This review considers the current results in thyroid cancer biomarker research with a focus on metabolomics

Effects of proteolytic digestion on the cyanotoxins microcystin-LR and cylindrospermopsin: the importance in integrating the bioaccessibility in human health risk assessment

The occurrence and proliferation of toxic cyanobacterial blooms are an emergent environmental concern worldwide. Microcystin-LR (MC-LR), a potent hepatotoxin, is the most documented and studied cyanotoxin. The cytotoxin cylindrospermopsin (CYN) has been recognized of increased concern due to the invasive nature of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that edible aquatic organisms, especially bivalves, can accumulate high levels of these cyanotoxins. MC-LR and CYN are stable at a wide range of temperatures and pHs, thus the knowledge of the influence of human digestion on its concentration in food is required to achieve a more accurate health risk assessment. The aim of this study was to assess the MC-LR and CYN bioaccessibility in edible bivalves. Clams (C. fluminea) fed MC-LR-producing M. aeruginosa and mussels (M. galloprovincialis) fed CYN-producing C. raciborskii were subjected to an in vitro digestion model adapted from Maulvault et al. (2011) and Versantvoort et al. (2005). Bioaccessibility of MC-LR and CYN were then assessed by LC-MS/MS. The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of its degradation by pancreatic enzymes. The in vitro digestion with salivary and gastrointestinal juices considerably decreased the CYN availability in uncooked and steamed mussels. Our results suggest that risk assessment based on MC-LR and CYN concentration in raw products might not be representative of true human exposure, once bioaccessibility strongly reduces the potential toxicological risks. Thus, the incorporation of the bioaccessibility of these cyanotoxins in the human exposure estimation would be of particular relevance to the application of more forceful management measures.info:eu-repo/semantics/publishedVersio

Circulating biomarkers in schizophrenia: a proteomics perspective.

info:eu-repo/semantics/publishedVersio

Proteomics Reveals mRNA Regulation and the Action of Annexins in Thyroid Cancer

Differentiated thyroid cancer is the most common malignancy of the endocrine system. Although most thyroid nodules are benign, given the high incidence of thyroid nodules in the population, it is important to understand the differences between benign and malignant thyroid cancer and the molecular alterations associated with malignancy to improve detection and signal potential diagnostic, prognostic, and therapeutic targets. Proteomics analysis of benign and malignant human thyroid tissue largely revealed changes indicating modifications in RNA regulation, a common cancer characteristic. In addition, changes in the immune system and cell membrane/endocytic processes were also suggested to be involved. Annexin A1 was considered a potential malignancy biomarker and, similarly to other annexins, it was found to increase in the malignant group. Furthermore, a bioinformatics approach points to the transcription factor Sp1 as being potentially involved in most of the alterations seen in the malignant thyroid nodules

Partitioning the Proteome: Phase Separation for Targeted Analysis of Membrane Proteins in Human Post-Mortem Brain

Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain

Disruption of the sea bass skin-scale barrier by antidepressant fluoxetine and estradiol: in vivo and in vitro evidence

Trabajo presentado en la Joint 30th Conference of the European Society for Comparative Endocrinology and of the 9th International Society for Fish Endocrinology, celebrada en Faro (Portugal) del 04 al 08 de septiembre de 2022.Fluoxetine (FLX) is a highly prescribed selective inhibitor of serotonin-reuptake and an emerging pollutant affecting fish behaviour, stress and reproduction, but little is known about possible actions and mechanisms in barrier tissues. We combined in vivo and in vitro approaches to demonstrate multi-level impacts of FLX on the sea bass (Dicentrarchus labrax) skin-scale barrier and on the estrogenic system. Juvenile sea bass intraperitoneally injected with FLX had significantly increased levels of FLX and its metabolite nor-FLX. In contrast to the natural estrogen E2, FLX did not increase plasma calcium, phosphorus (P) or vitellogenin, although a slight decrease in scale P content was detected. Quantitative SWATH-MS proteomics of the scales identified 134 proteins that were affected by FLX. Modified proteins were mainly related to extracellular matrix and protein turnover and energy production, 31 of which were also affected by E2. Multiple estrogen receptors and genes related to serotonin activity, transport and degradation were expressed in sea bass scales and transcript abundance of some of them was modulated by E2 and/or FLX. Using a minimally invasive in vitro bioassay with cultured sea bass scales and adhering epithelia we showed direct effects of FLX exposure on enzymatic activity associated with mineral mobilization, while the expression of estrogen receptors was not significantly affected. In in vitro receptor-reporter assays, FLX alone did not activate any of the three sea bass nuclear estrogen receptors but had antiestrogenic effects on Esr1/2b when in co-treatment with E2, and directly activated both plasma membrane Gprotein-coupled estrogen receptors. The combination of in vitro and in vivo assays substantiated the notion that FLX disrupted scale physiology through several different processes, with probable consequences for fish health, and revealed that some of the mechanisms of disruption can result from direct interaction with multiple estrogen .Projects UIDB/04326/2020, PTDC/AAG-GLO/4003/2012 and DL57/2016/CP1361/CT0015 from FCT (Pt); EU Interreg FR-UK project RedPol; grant AGL2015-67477-C2-1- R (Sp)

The Enhanced Efficacy of Intracellular Delivery of Doxorubicin/C6-Ceramide Combination Mediated by the F3 Peptide/Nucleolin System Is Supported by the Downregulation of the PI3K/Akt Pathway

Targeting multiple cellular populations is of high therapeutic relevance for the tackling of solid tumors heterogeneity. Herein, the ability of pegylated and pH-sensitive liposomes, functionalized with the nucleolin-binding F3 peptide and containing doxorubicin (DXR)/C6-ceramide synergistic combination, to target, in vitro, ovarian cancer, including ovarian cancer stem cells (CSC), was assessed. The underlying molecular mechanism of action of the nucleolin-mediated intracellular delivery of C6-ceramide to cancer cells was also explored. The assessment of overexpression of surface nucleolin expression by flow cytometry was critical to dissipate differences identified by Western blot in membrane/cytoplasm of SKOV-3, OVCAR-3 and TOV-112D ovarian cancer cell lines. The former was in line with the significant extent of uptake into (bulk) ovarian cancer cells, relative to non-targeted and non-specific counterparts. This pattern of uptake was recapitulated with putative CSC-enriched ovarian SKOV-3 and OVCAR-3 sub-population (EpCAMhigh/CD44high). Co-encapsulation of DXR:C6-ceramide into F3 peptide-targeted liposomes improved cytotoxic activity relative to liposomes containing DXR alone, in an extent that depended on the intrinsic resistance to DXR and on the incubation time. The enhanced cytotoxicity of the targeted combination was mechanistically supported by the downregulation of PI3K/Akt pathway by C6-ceramide, only among the nucleolin-overexpressing cancer cells presenting a basal p-Akt/total Akt ratio lower than 1

Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cells secretome?

Introduction: The use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear. Methods: In the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells. Results: The present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome. Conclusions: Our results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications. © 2015 Teixeira et al.Portuguese Foundation for Science and Technology (FCT) (Ciência 2007 program and IF Development Grant (AJS); and pre-doctoral fellowships to FGT (SFRH/69637/ 2010) and SIA (SFRH/BD/81495/2011); Canada Research Chairs (LAB) and a SSE Postdoctoral Fellowship (KMP); The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005); co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER).info:eu-repo/semantics/publishedVersio

In vitro dissolution kinetic for mycophenolic acid derivatives tablets

Mycophenolate mofetil (MMF) and mycophenolate sodium (MPS) are an ester and a salt of mycophenolic acid. They have different kinetic in vivo characteristics due to differences in molecular structures, physicochemical properties and formulations administered. In this study, dissolution profiles of reference products were tested in different media to evaluate the effect of pH, kinetic dissolution and the best statistical model that can be used to predict the release of both drugs. The drug release was determined by using a validated ultraviolet spectrophotometry method, λ 250 nm. The method showed to be selective, linear, precise and accurate for MMF in 0.1 M HCl and MPS in sodium phosphate buffer pH 6.8. Dissolution kinetics models of zero order, first order, Higuchi, Hixson-Crowell and Weibull were applied to data in order to select the best fit by linear regression. The regression parameters were estimated and the models were evaluated with the results of residuals and coefficient of determination. The residuals obtained from dissolution kinetics models were random, uncorrelated, and normally distributed with constant variance. The R² values (74.7% for MMF and 95.8% for MPS) demonstrated good ability of the Weibull regression to explain the variability and to predict the drugs' release

Biopharmaceutics classification system: importance and inclusion in biowaiver guidance

Pharmacological therapy is essential in many diseases treatment and it is important that the medicine policy is intended to offering safe and effective treatment with affordable price to the population. One way to achieve this is through biowaiver, defined as the replacement of in vivo bioequivalence studies by in vitro studies. For biowaiver of new immediate release solid oral dosage forms, data such as intestinal permeability and solubility of the drug are required, as well as the product dissolution. The Biopharmaceutics Classification System (BCS) is a scientific scheme that divides drugs according to their solubility and permeability and has been used by various guides as a criterion for biowaiver. This paper evaluates biowaiver application, addressing the general concepts and parameters used by BCS, making a historical account of its use, the requirements pertaining to the current legislation, the benefits and risks associated with this decision. The results revealed that the use of BCS as a biowaiver criterion greatly expands the therapeutics options, contributing to greater therapy access of the general population with drug efficacy and safety guaranteed associated to low cost