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      Characterization of a new type of neuronal 5-HT G- protein coupled receptor in the cestode nervous system

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          Abstract

          Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae ( syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT 1a. Functional heterologous expression studies showed that Eca-5-HT 1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT 1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT 1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT 1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.

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          MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms.

          The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
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            The Phyre2 web portal for protein modeling, prediction and analysis.

            Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.
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              CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP.

              The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Investigation
                Role: Data curationRole: InvestigationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 November 2021
                2021
                : 16
                : 11
                : e0259104
                Affiliations
                [1 ] Laboratorio de Toxinopatología, Centro de Patología Experimental y Aplicada, Facultad de Medicina, Universidad de Buenos Aires (UBA), Ciudad Autónoma de Buenos Aires, Argentina
                [2 ] Instituto de Investigaciones en Microbiología y Parasitología Médica (IMPaM, UBA-CONICET), Facultad de Medicina (FMed), Universidad de Buenos Aires (UBA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
                [3 ] Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Ciudad Autónoma de Buenos Aires, Argentina
                [4 ] Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States of America
                [5 ] Departamento de Parasitología, INEI-ANLIS, “Dr Carlos G. Malbrán”, Ciudad Autónoma de Buenos Aires, Argentina
                [6 ] Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
                Universitat Leipzig, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0003-1406-0357
                https://orcid.org/0000-0002-5395-8402
                https://orcid.org/0000-0002-0520-0226
                https://orcid.org/0000-0003-0977-9666
                https://orcid.org/0000-0002-4725-541X
                https://orcid.org/0000-0002-9699-4964
                https://orcid.org/0000-0001-6592-0877
                https://orcid.org/0000-0002-5271-6321
                Article
                PONE-D-21-12202
                10.1371/journal.pone.0259104
                8584985
                d0f079e5-8082-4c36-b222-0b6ab40817b6
                © 2021 Camicia et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 April 2021
                : 12 October 2021
                Page count
                Figures: 5, Tables: 3, Pages: 28
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01 AI145871
                Award Recipient :
                Funded by: Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET)
                Award ID: PICT 2017 N 2966
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100010253, Secretaría de Ciencia y Técnica, Universidad de Buenos Aires;
                Award ID: 20020150100160BA
                This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina and by Secretaria de Ciencia y Técnica (UBACyT), Universidad de Buenos Aires, Facultad de Medicina, Argentina. Project Programación Científica 2016, code 20020150100160BA, PICT 2017 N°2966. HRV is a recipient of a CONICET postdoctoral fellowship. JSM and SKP were supported by the NIH (R01 AI145871). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Neurochemistry
                Neurotransmitters
                Biogenic Amines
                Serotonin
                Biology and Life Sciences
                Neuroscience
                Neurochemistry
                Neurotransmitters
                Biogenic Amines
                Serotonin
                Biology and Life Sciences
                Biochemistry
                Proteins
                Transmembrane Receptors
                G Protein Coupled Receptors
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Transmembrane Receptors
                G Protein Coupled Receptors
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Helminths
                Cestodes
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                Zoology
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                Invertebrates
                Helminths
                Cestodes
                Biology and Life Sciences
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                Eukaryota
                Animals
                Invertebrates
                Flatworms
                Cestodes
                Biology and Life Sciences
                Zoology
                Animals
                Invertebrates
                Flatworms
                Cestodes
                Biology and Life Sciences
                Biochemistry
                Proteins
                Transmembrane Receptors
                Serotonin Receptors
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Transmembrane Receptors
                Serotonin Receptors
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                Biochemistry
                Proteins
                Transmembrane Receptors
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                Cell Biology
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                Molecular Biology
                Molecular Biology Techniques
                Cloning
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                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Physical Sciences
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                Chemical Compounds
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Custom metadata
                All the accession numbers are available from the Genbank database (accession number(s) MW535743 for Eca-5HT1a; MW535744 for Mvo-5HT1a and MW535745 for Hmi-5HT1a).

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