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      Neuropeptidergic Signaling in the American Lobster Homarus americanus: New Insights from High-Throughput Nucleotide Sequencing

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          Abstract

          Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

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          DiANNA: a web server for disulfide connectivity prediction

          Correctly predicting the disulfide bond topology in a protein is of crucial importance for the understanding of protein function and can be of great help for tertiary prediction methods. The web server outputs the disulfide connectivity prediction given input of a protein sequence. The following procedure is performed. First, PSIPRED is run to predict the protein's secondary structure, then PSIBLAST is run against the non-redundant SwissProt to obtain a multiple alignment of the input sequence. The predicted secondary structure and the profile arising from this alignment are used in the training phase of our neural network. Next, cysteine oxidation state is predicted, then each pair of cysteines in the protein sequence is assigned a likelihood of forming a disulfide bond—this is performed by means of a novel architecture (diresidue neural network). Finally, Rothberg's implementation of Gabow's maximum weighted matching algorithm is applied to diresidue neural network scores in order to produce the final connectivity prediction. Our novel neural network-based approach achieves results that are comparable and in some cases better than the current state-of-the-art methods.
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            InterPro--an integrated documentation resource for protein families, domains and functional sites.

            InterPro is a new integrated documentation resource for protein families, domains and functional sites, developed initially as a means of rationalising the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Merged annotations from PRINTS, PROSITE and Pfam form the InterPro core. Each combined InterPro entry includes functional descriptions and literature references, and links are made back to the relevant parent database(s), allowing users to see at a glance whether a particular family or domain has associated patterns, profiles, fingerprints, etc. Merged and individual entries (i.e. those that have no counterpart in the companion resources) are assigned unique accession numbers. Release 1.2 of InterPro (June 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification (PTMs) encoded by 6581 different regular expressions, profiles, fingerprints and Hidden Markov Models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1000000 hits from 264333 different proteins out of 384572 in SWISS-PROT and TrEMBL).
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              The unique evolution of neuropeptide genes in the silkworm Bombyx mori.

              Cloning-based approach combining homology search in the Bombyx genome sequence and Rapid Amplification of cDNA Ends (RACE) resulted in annotation of 23 neuropeptide genes and different splicing variants of three genes. In total 37 neuropeptide genes in addition to bombyxin gene family have been identified in Bombyx. Comparison of available insect neuropeptide genes revealed that the silkworm genome contains most conserved neuropeptide genes except those encoding proctolin, vasopressin-like peptide and neuropeptide-like precursor 2. In addition, we identified several paralogous neuropeptide genes which have not been found in other insects. The Bombyx genome contains a triplet of paralogous genes encoding adipokinetic hormones (AKH), two genes encoding different neuropeptide Fs (NPFs) and a tandem of related SIFamide and IMFamide genes. A novel gene coding for CCHamide was cloned and its expression in the CNS and midgut was demonstrated. Differential splicing was observed for the first time in transcripts for diuretic hormones and cardioacceleratory peptides 2b. Most paralogous genes or splicing variants of the same gene showed different expression patterns in the central nervous system (CNS). These results suggest that unique duplication and differential expression of several neuropeptide genes occurred during the evolution in Bombyx. This may be an effective mechanism for functional diversification of conserved neuropeptides.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                30 December 2015
                2015
                : 10
                : 12
                : e0145964
                Affiliations
                [1 ]Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center and Technology, 6500 College Station, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, Hawaii, 96822, United States of America
                [2 ]Department of Biology, Bowdoin College, 6500 College Station, Brunswick, Maine, 04672, United States of America
                [3 ]Division of Biological Sciences, University of Missouri, 218A LeFevre Hall, Columbia, Missouri, 65211, United States of America
                Wake Forest University, UNITED STATES
                Author notes

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

                Conceived and designed the experiments: AEC DJS PSD. Performed the experiments: AEC MC TJL MGP DNS MES DJS PSD. Analyzed the data: AEC MC TJL MGP DNS MES DJS PSD. Wrote the paper: AEC DJS PSD.

                Article
                PONE-D-15-50409
                10.1371/journal.pone.0145964
                4696782
                26716450
                b5fb9184-2dbb-4781-b2b5-aeaff9754af0
                © 2015 Christie 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
                : 18 November 2015
                : 10 December 2015
                Page count
                Figures: 3, Tables: 3, Pages: 29
                Funding
                Financial support for this study was provided by the National Science Foundation (IOS-1353023 to A.E.C and IOS-1354567 to P.S.D), the National Institutes of Health (Maine IDeA Networks of Biomedical Research Excellence (INBRE) [P20GM0103423 from NIGMS to Dr. Patricia Hand, Mount Desert Island Biological Laboratory]), the Doherty Fund of Bowdoin College (to D.N.S.), the American Physiological Society (to M.E.S.), and the Cades Foundation of Honolulu, Hawaii (to A.E.C). The cost of transcriptome sequencing was supported by the National Institutes of Health (MH46742 to Eve Marder, Brandeis University). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                This Transcriptome Shotgun Assembly (TSA) project has been deposited at GenBank under the Accession No. GEBG00000000 (BioProject No. PRJNA300643; BioSample No. SAMN04230440). The version described in this paper is the first version, GEBG01000000.

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