A Transcriptomic Approach to the Recruitment of Venom Proteins in a Marine Annelid

CEECIND/02699/2017The growing number of known venomous marine invertebrates indicates that chemical warfare plays an important role in adapting to diversified ecological niches, even though it remains unclear how toxins fit into the evolutionary history of these animals. Our case study, the Polychaeta Eulalia sp., is an intertidal predator that secretes toxins. Whole-transcriptome sequencing revealed proteinaceous toxins secreted by cells in the proboscis and delivered by mucus. Toxins and accompanying enzymes promote permeabilization, coagulation impairment and the blocking of the neuromuscular activity of prey upon which the worm feeds by sucking pieces of live flesh. The main neurotoxins ("phyllotoxins") were found to be cysteine-rich proteins, a class of substances ubiquitous among venomous animals. Some toxins were phylogenetically related to Polychaeta, Mollusca or more ancient groups, such as Cnidaria. Some toxins may have evolved from non-toxin homologs that were recruited without the reduction in molecular mass and increased specificity of other invertebrate toxins. By analyzing the phylogeny of toxin mixtures, we show that Polychaeta is uniquely positioned in the evolution of animal venoms. Indeed, the phylogenetic models of mixed or individual toxins do not follow the expected eumetazoan tree-of-life and highlight that the recruitment of gene products for a role in venom systems is complex.publishersversionpublishe

Comprehensive review on the interaction between natural compounds and brain receptors: Benefits and toxicity

Given their therapeutic activity, natural products have been used in traditional medicines throughout the centuries. The growing interest of the scientific community in phytopharmaceuticals, and more recently in marine products, has resulted in a significant number of research efforts towards understanding their effect in the treatment of neurodegenerative diseases, such as Alzheimer's (AD), Parkinson (PD) and Huntington (HD). Several studies have shown that many of the primary and secondary metabolites of plants, marine organisms and others, have high affinities for various brain receptors and may play a crucial role in the treatment of diseases affecting the central nervous system (CNS) in mammalians. Actually, such compounds may act on the brain receptors either by agonism, antagonism, allosteric modulation or other type of activity aimed at enhancing a certain effect. The current manuscript comprehensively reviews the state of the art on the interactions between natural compounds and brain receptors. This information is of foremost importance when it is intended to investigate and develop cutting-edge drugs, more effective and with alternative mechanisms of action to the conventional drugs presently used for the treatment of neurodegenerative diseases. Thus, we reviewed the effect of 173 natural products on neurotransmitter receptors, diabetes related receptors, neurotrophic factor related receptors, immune system related receptors, oxidative stress related receptors, transcription factors regulating gene expression related receptors and blood-brain barrier receptors.The author A.R.S. is grateful to the authors C.G. and J.M.R for the scientific assistance and suggestions shared throughout the supervision of her B.Sc. final project. The author A.R.S. also acknowledges the Department of Biology (DB) and the Centro de Biologia Molecular e Ambiental [Centre of Molecular and Environmental Biology] (CBMA) from School of Sciences (EC), University of Minho (UM), Braga, Portugal, by providing all the conditions leading to the B.Sc. in Biochemistry. The corresponding author C.G. is grateful to Fundação para a Ciência e a Tecnologia [Foundation for Science and Technology, FCT I.P.] (FCT I.P.) for the FCT Investigator (IF/01332/2014/CP1255/CT0001). The author J.M.R. acknowledges the CBMA and the Instituto de Ciência e Inovação para a Biosustentabilidade [Institute of Science and Innovation for Biosustainability] (IBS) from University of Minho (UM), Braga, Portugal, where he recently carried out his activities as invited assistant researcher and professor. The author J.M.R. is also grateful to the Laboratório Associado para a Química Verde (LAQV) [Green Chemistry Laboratory] from REQUIMTE – Rede de Química e Tecnologia [REQUIMTE – Chemistry and Technology Network], as well as to the Department of Chemistry and Biochemistry (DCB) from the Faculty of Sciences from University of Porto (FCUP), Porto, Portugal, where he is currently researcher. Regarding to the author J.M.R., this work was supported by the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) (Research project entitled “EcoAgriFood: Innovative green products and processes to promote Agri-Food BioEconomy”) and PTDC/SAUNUT/30448/2017 (POCI-01-0145-FEDER-030448) (Research project entitled “Poly4CD: Action of Dietary Polyphenols in Preventing Celiac Disease”) funded by Portuguese national funds through Fundação para a Ciência e Tecnologia [Foundation for Science and Technology] (FCT-I.P.)/Ministério da Ciência, Tecnologia e Ensino Superior [Ministry of Science, Technology and Higher Education] (MCTES), and Fundo Europeu de Desenvolvimento Regional [European Regional Development Fund] (FEDER), under the scope of the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) [COMPETE2020 – Competitiveness and Internationalization Operational Program] and by COST Action 18101 “SOURDOMICS”, supported by COST (European Cooperation in Science and Technology). C. G. and C. D.-M. are grateful for the financial support from FCT/MCTES through national funds (UID/QUI/50006/2019). C. G. and C. D.-M. would also like to thank the EU and FCT for funding through the projects: DESignBIOtecHealth - New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases (Portugal2020, Norte-01-0145-FEDER-000024) and project PTDC/OCE-ETA/30240/2017- SilverBrain - From sea to brain: Green neuroprotective extracts for nanoencapsulation and functional food production (POCI-01-0145-FEDER-030240); and to the REQUIMTE for the project “Sea_4_Brain_Food”. All the authors acknowledge the CBMA and IB-S by the financial support provided specifically for this open access publication.info:eu-repo/semantics/publishedVersio

Genetic predisposition for aggressive behaviour related with dopamine and serotonin pathways : an overview

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio

Specific Antiproliferative Properties of Proteinaceous Toxin Secretions from the Marine Annelid Eulalia sp. onto Ovarian Cancer Cells

The Portuguese Foundation for Science and Technology (FCT) funded project WormALL (PTDC/BTA-BTA/28650/2017) plus the grants SFRH/BD/109462/2015 to A.P.R., and CEECIND/02699/2017 to A.R.G. The Applied Molecular Biosciences Unit-UCIBIO is financed by national funds from FCT, ref. UIDB/04378/2020. FCT, along with the European Regional Development Fund (ERDF) through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation also funded the projects: POCI-01-0145-FEDER-007440 (UID/NEU/04539/2019), POCI-01-0145-FEDER-016428 (SAICTPAC/0010/2015), POCI-01-0145-FEDER-029311 (PTDC/BTM-TEC/29311/2017), POCI-01-0145-FEDER-30943 (PTDC/MEC-PSQ/30943/2017) and PTDC/MED-NEU/27946/2017. The work was also funded by the National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125 (ref.: ROTEIRO/0028/2013).As Yondelis joins the ranks of approved anti-cancer drugs, the benefit from exploring the oceans' biodiversity becomes clear. From marine toxins, relevant bioproducts can be obtained due to their potential to interfere with specific pathways. We explored the cytotoxicity of toxin-bearing secretions of the polychaete Eulalia onto a battery of normal and cancer human cell lines and discovered that the cocktail of proteins is more toxic towards an ovarian cancer cell line (A2780). The secretions' main proteins were identified by proteomics and transcriptomics: 14-3-3 protein, Hsp70, Rab3, Arylsulfatase B and serine protease, the latter two being known toxins. This mixture of toxins induces cell-cycle arrest at G2/M phase after 3h exposure in A2780 cells and extrinsic programmed cell death. These findings indicate that partial re-activation of the G2/M checkpoint, which is inactivated in many cancer cells, can be partly reversed by the toxic mixture. Protein-protein interaction networks partake in two cytotoxic effects: cell-cycle arrest with a link to RAB3C and RAF1; and lytic activity of arylsulfatases. The discovery of both mechanisms indicates that venomous mixtures may affect proliferating cells in a specific manner, highlighting the cocktails' potential in the fine-tuning of anti-cancer therapeutics targeting cell cycle and protein homeostasis.publishersversionpublishe

The genetic susceptibility linking preterm birth and periodontal disease : a review

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio

Aerocyte specification and lung adaptation to breathing is dependent on alternative splicing changes

© 2022 Fidalgo et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found that Vegfa switches from the Vegfa 164 isoform to the longer Vegfa 188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type-specific regulation of Vegfa alternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.This work was supported by European Research Council (ERC starting grant [679368]), the European Union (H2020-TWINN-2015 – Twinning [692322]), Fundação para a Ciência e Tecnologia (FCT) (PTDC/MED-PAT/31639/2017, and UIDP/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO), and Fondation Leducq (17CVD03). CG Fonseca was supported by a PhD fellowship from the doctoral program Bioengineering: Cellular Therapies and Regenerative Medicine funded by Fundação para a Ciência e Tecnologia (FCT) (PD/BD/128375/2017). T Balboni was supported by a PhD fellowship from the doctoral program “Oncology, Hematology and Pathology - 30th Cycle” funded by University of Bologna, Italy. P Caldas was supported by a postdoctoral researcher fellowship from FCT (PTDC/MED-ONC/28660/2017). AASF Raposo was supported by FCT and Fundo Europeu de Desenvolvimento Regional (FEDER) PAC-PRECISE-LISBOA-01-0145-FEDER-016394 and by an assistant researcher contract from FCT (CEECIND/01474/2017). AR Grosso was supported by a principal investigator contract from FCT (CEECIND/02699/2017). FF Vasconcelos was supported by a postdoctoral researcher contract from FCT (CEECIND/04251/2017). CA Franco was supported by a principal investigator contract from FCT (CEECIND/02589/2018).info:eu-repo/semantics/publishedVersio

Sialyl LewisX/A and Cytokeratin Crosstalk in Triple Negative Breast Cancer

project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. Publisher Copyright: © 2023 by the authors.Triple-negative breast cancer (TNBC) encompasses multiple entities and is generally highly aggressive and metastatic. We aimed to determine the clinical and biological relevance of Sialyl-Lewis X and A (sLeX/A)—a fucosylated glycan involved in metastasis—in TNBC. Here, we studied tissues from 50 TNBC patients, transcripts from a TNBC dataset from The Cancer Genome Atlas (TCGA) database, and a primary breast cancer cell line. All 50 TNBC tissue samples analysed expressed sLeX/A. Patients with high expression of sLeX/A had 3 years less disease-free survival than patients with lower expression. In tissue, sLeX/A negatively correlated with cytokeratins 5/6 (CK5/6, which was corroborated by the inverse correlation between fucosyltransferases and CK5/6 genes. Our observations were confirmed in vitro when inhibition of sLeX/A remarkably increased expression of CK5/6, followed by a decreased proliferation and invasion capacity. Among the reported glycoproteins bearing sLeX/A and based on the STRING tool, α6 integrin showed the highest interaction score with CK5/6. This is the first report on the sLeX/A expression in TNBC, highlighting its association with lower disease-free survival and its inverse crosstalk with CK5/6 with α6 integrin as a mediator. All in all, sLeX/A is critical for TNBC malignancy and a potential prognosis biomarker and therapeutic target.publishersversionpublishe

Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

PTDC/BIA-MOL/30438/2017 PTDC/MED-OUT/4301/2020 RiboMed 857119 PD/BD/128292/2017 LCF/PR/HP21/52310016 PTDC/BIA-MOL/6624/2020 PTDC/MED-ONC/7864/2020DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in the absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops shows a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.publishersversionpublishe

Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ T-Cells

BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials

TCR signal strength controls thymic differentiation of discrete proinflammatory gamma delta T cell subsets

The mouse thymus produces discrete gd T cell subsets that make either interferon-g (IFN-g) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g+/− Cd3d+/− (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on gd T cells. CD3DH mice had normal numbers and phenotypes of ab thymocyte subsets, but impaired differentiation of fetal Vg6+ (but not Vg4+) IL-17- producing gd T cells and a marked depletion of IFN-g-producing CD122+ NK1.1+ gd T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-g+ gd T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory gd T cell subsets and their impact on pathophysiology