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Anjos Guerreiro, Liliana Isabel Tomé

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Now showing 1 - 9 of 9
  • Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts
    Publication . Letsiou, Sophia; Félix, Rute; Cardoso, João CR; L, Anjos; Mestre, Ana L G; H, Gomes; Power, Deborah
    Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein's structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein's biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
  • Deciphering the role of cartilage protein 1 in human dermal fibroblasts: a transcriptomic approach
    Publication . Letsiou, Sophia; Manchado, Manuel; Zografaki, Mariela; Marka, Sofia; L, Anjos; Skliros, Dimitrios; Martínez-Blanch, Juan F.; Flemetakis, E.; Power, Deborah
    Cartilage acidic protein 1A (hCRTAC1-A) is an extracellular matrix protein (ECM) of human hard and soft tissue that is associated with matrix disorders. The central role of fibroblasts in tissue integrity and ECM health made primary human dermal fibroblasts (NHDF) the model for the present study, which aimed to provide new insight into the molecular function of hCRTAC1-A. Specifically, we explored the differential expression patterns of specific genes associated with the presence of hCRTAC1-A by RNA-seq and RT-qPCR analysis. Functional enrichment analysis demonstrated, for the very first time, that hCRTAC1-A is involved in extracellular matrix organization and development, through its regulatory effect on asporin, decorin, and complement activity, in cell proliferation, regeneration, wound healing, and collagen degradation. This work provides a better understanding of putative hCRTAC1-A actions in human fibroblasts and a fundamental insight into its function in tissue biology.
  • Dilution of seawater affects the Ca2 + transport in the outer mantle epithelium of crassostrea gigas
    Publication . Sillanpää, J. Kirsikka; Cardoso, João CR; Félix, Rute; Anjos, Liliana; Power, Deborah; Sundell, Kristina
    Varying salinities of coastal waters are likely to affect the physiology and ion transport capabilities of calcifying marine organisms such as bivalves. To investigate the physiological effect of decreased environmental salinity in bivalves, adult oysters (Crassostrea gigas) were exposed for 14 days to 50% seawater (14) and the effects on mantle ion transport, electrophysiology and the expression of Ca2+ transporters and channels relative to animals maintained in full strength sea water (28) was evaluated. Exposure of oysters to a salinity of 14 decreased the active mantle transepithelial ion transport and specifically affected Ca2+ transfer. Gene expression of the Na+/K+-ATPase and the sarco(endo)plasmic reticulum Ca2+-ATPase was decreased whereas the expression of the T-type voltage-gated Ca channel and the Na+/Ca2+-exchanger increased compared to animals maintained in full SW. The results indicate that decreased environmental salinities will most likely affect not only osmoregulation but also bivalve biomineralization and shell formation.
  • Proteomics of fish white muscle and Western blotting to detect putative allergens
    Publication . Anjos, Liliana; Loukissas, A. Ζ.; Power, Deborah
    A detailed workflow is provided for preparation from teleost fish white muscle of extracts for proteomics analysis. The protocol generates samples that can be analyzed by SWATH (Sequential Window data independent Acquisition of the Total High-resolution-Mass Spectra), a modern MS-based quantitative label free technology. The main steps for the extraction of three independent protein fractions, (1) soluble sarcoplasmic, (2) soluble myofibrillar, and (3) insoluble material, from fish white muscle are detailed. Coupled to the protein extraction protocol a Western blotting approach is outlined for detection of common fish allergens, in this case β-parvalbumin, in the white muscle sarcoplasmic protein fraction.
  • Cartilage acidic protein a novel therapeutic factor to improve skin damage repair?
    Publication . Félix, Rute; Anjos, Liliana; Costa, Rita; Letsiou, Sophia; Power, Deborah Mary
    Fish skin has been gaining attention due to its efficacy as a human-wound-treatment product and to identify factors promoting its enhanced action. Skin fibroblasts have a central role in maintaining skin integrity and secrete extra cellular matrix (ECM) proteins, growth factors and cytokines to rapidly repair lesions and prevent further damage or infection. The effects on scratch repair of the ubiquitous but poorly characterized ECM protein, cartilage acidic protein 1 (CRTAC1), from piscine and human sources were compared using a zebrafish SJD.1 primary fibroblast cell line. A classic in vitro cell scratch assay, immunofluorescence, biosensor and gene expression analysis were used. Our results demonstrated that the duplicate sea bass Crtac1a and Crtac1b proteins and human CRTAC-1A all promoted SJD.1 primary fibroblast migration in a classic scratch assay and in an electric cell impedance sensing assay. The immunofluorescence analysis revealed that CRTAC1 enhanced cell migration was most likely caused by actin-driven cytoskeletal changes and the cellular transcriptional response was most affected in the early stage (6 h) of scratch repair. In summary, our results suggest that CRTAC1 may be an important factor in fish skin promoting damage repair.
  • Extracting protein from microalgae (Tetraselmis chuii) for proteome analysis
    Publication . L, Anjos; Estêvão, J.; Infante, Carlos; Mantecón, Lalia; Power, Deborah Mary
    Microalgae have high potential as a resource for sustainable and green protein for food or bioactive molecules. Nonetheless, despite the high protein content of microalgae (40 - 70% dry weight) progress in the characterization of their protein composition remains challenging. This is due to the highly variable chemical composition of microalgae strains and factors such as their rigid thick cell wall, polysaccharide content, protein stability, pH. The method described herein was developed to optimize protein extraction for proteome analysis of microalgae (Tetraselmis chuii) biomass. The effects on protein solubility of solvent type (organic, denaturing, and non-denaturing) combined with three customized microalgae disruption methods were investigated. The proteome targeted high quality protein extracts were for hydro-soluble proteins recovered by cell disruption using bead milling coupled to centrifugation (protein yield approximate to 13%). The developed method is inexpensive, efficient (yielding high-quality protein extracts with a low content of interfering compounds) and from an industrial perspective easily scalable and compatible with other applications. To add value to the end product we additionally propose the use of stabilizing agents to maintain protein solubility during refrigerated storage and a method targeting the fractionation of low molecular weight proteins. An inexpensive easy-to-do 5 step protocol for microalgae protein extracts. A protein extraction method free from dangerous or highly polluting chemicals. Production of high yield aqueous protein extracts suitable for proteomics. (C) 2022 The Authors. Published by Elsevier B.V.
  • Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiol
    Publication . L, Anjos; PI Pinto, PPinto; Santos, Soraia; Estêvão, M. Dulce; Santa, Cátia; Manadas, Bruno; Canario, A.V.M.; Power, Deborah
    Contamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17 beta-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent a cquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
  • Comparing the response of Citrus ×limon and Citrus ×sinensis to Trioza erytreae infestation using a proteomic approach
    Publication . Magalhães, Tomás; Dandlen, Susana; L, Anjos; Power, Deborah; Pereira, José Alberto; Duarte, Amilcar; Marques, Natália
    Citrus production is on high alert because of the devastating disease Huanglongbing (HLB), caused by the bacteria Candidatus Liberibacter spp.. With no viable treatment, current management practices rely on the control of its vectors, such as the African citrus psyllid,Trioza erytreae (Del Guercio 1918), which is already in the Mediterranean region (Portugal and north of Spain). This vector develops better in some citrus hosts, with Citrus ×limon described as the preferred host. To better understand the molecular response of citrus hosts to the psyllid, the phloem proteome of lemon (Citrus ×limon) and orange (Citrus ×sinensis) plants infested with T. erytreae was compared with equivalent non infested plants. Infestation was established with isolated plants by exposing them to 10 T. erytreae adults. Nymphs of T. erytreae at the 4-5th instar stage were removed from plants and infested leaf phloem was extracted. In control plants phloem was extracted from leaves of a similar size and developmental stage. The experiment was done under controlled conditions of temperature, light and humidity. Phloem was analyzed by nanoLC-MS/MS. A total of 48 and 1265 differentially abundant proteins (DAP) were identified in lemon and orange plants, respectively, with 18 proteins common to both species. The topmost enriched GO terms retrieved for upregulated proteins in lemon plants were assigned to organic acid and cellular amino acid metabolic processes. The topmost enriched GO terms in orange plants included organonitrogen compound metabolic process, cellular component assembly, establishment of protein localization, while downregulated terms were associated with carbohydrate metabolic process. This study revealed that T. erytreae infestation promoted distinct modifications in the phloem proteome of lemon and orange plants. This work is part of a group of studies that focus on this insect-plant interaction that aims for more informed and improved T. erytreae control.
  • Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish fillets
    Publication . Pinto, Patricia IS; NAJAFPOUR, BABAK; Lima, Pedro; P. Machado; Aires, Tania; Engelen, Aschwin; Tsironi, T.; Anjos Guerreiro, Liliana Isabel Tomé; Power, Deborah Mary
    Fresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7-14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.